CLINICAL BACTERIOLOGY

brainrotgaIore || May 2024

OUTLINE ◘ GRAM POSITIVE NON-SPORE FORMING BACILLI

 Listeria monocytogenes
◘ BACTERIAL TAXONOMY AND NOMENCLATURE  Erysipelothrix rhusiopathiae
◘ BACTERIAL CHARACTERISTICS  Lactobacillus
◘ BACTERIAL METABOLISM  Corynebacterium
◘ BACTERIAL PATHOGENECITY AND VIRULENCE 1. Corynebacterium diphtheria
◘ BACTERIAL GROWTH REQUIREMENTS 2. Other Corynebacterium
◘ BACTERIAL GENETICS  Kurthia
◘ METHODS OF MICROBIAL CONTROL  Rothia
◘ QUALITY CONTROL IN MICROBIOLOGY LABORATORY
◘ TYPES OF MICROSCOPY ◘ AEROBIC ACTINOMYCETES
◘ SPECIMEN, COLLECTION, TRANSPORT, AND PROCESSING  Nocardia spp.
◘ BACTERIAL IDENTIFICATION  Rhodococcus equi
◘ STUDY OF CULTURAL CHARACTERISTICS  Gardonia spp.
◘ ANTISUSCEPTIBILITY TEST (AST)  Streptomyces spp.
 Actinomadura spp.
◘ GRAM POSITIVE COCCI  Tropheryma whipplei
 Staphylococci
1. Staphylococcus aureus ◘ “ACID FAST” BACILLI
2. Coagulase-negative Staphylococci  Mycobacterium tuberculosis Complex
 Micrococci 1. Mycobacterium tuberculosis
 Streptococci 2. Mycobacterium bovis
1. Group A Streptococci: Streptococcus pyogenes 3. Mycobacterium canetti
2. Group B Streptococci: Streptococcus agalactiae 4. Mycobacterium africanum
3. Group C Streptococci 5. Mycobacterium microti
4. Group D Non-enterococci and Enterococci 6. Mycobacterium pinnipedii
5. Streptococcus pneumonia ( aka Diplococcus pneumonia, Pneumococci) 7. Mycobacterium caprae
6. Viridans Streptococci  Mycobacterium leprae
7. Nutritionally Variant Streptococci
 Other Gram Positive, Catalase Negative Organisms ◘ GRAM NEGATIVE FERMENTATIVE BACILLI
 Rapid Lactose Fermenters “Coliforms”
◘ GRAM NEGATIVE COCCI 1. Escherichia coli
 Neisseria 2. Klebsiella spp.
 Moraxella (Branhamella) 3. Enterobacter spp.
 Non-Lactose Fermenters “PPM Group”
◘ GRAM POSITIVE SPORE FORMING BACILLI 1. Proteus
 Bacillus 2. Providencia
1. Bacillus anthracis 3. Morganella
2. Bacillus cereus  Yersinia spp.
3. Bacillus subtilis 1. Yersinia pestis
4. Other Bacillus 2. Yersinia enterocolitica
 Clostridium 3. Yersinia pseudotuberculosis
1. Clostridium perfringens (aka Clostridium weichii)  Salmonella-Shigella
2. Clostridium tetani 1. Salmonella spp.
3. Clostridium botulinum 2. Shigella spp.
4. Clostridium difficile  Other Enterobacteriaceae
5. Clostridium septicum 1. Citrobacter
6. Clostridium ramosum 2. Serratia
3. Edwardsiella
4. Hafnia
5. Plesiomonas

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◘ GRAM NEGATIVE NON-ENTERICS ◘ ANAEROBIC BACTERIA
 Vibrionaceae  Gram Positive Anaerobic Cocci
1. Vibrio spp. 1. Peptococcus
2. Aeromonas spp. 2. Peptostreptococcus
3. Plesiomonas spp. 3. Sarcina
 Helicobacter  Gram Positive Anaerobic Rods
 Campylobacter 1. Non-Spore Forming
a. Actinomyces
◘ GRAM NEGATIVE NON-FERMENTATIVE BACILLI b. Bifidbacterium
 Family Pseudomonadaceae c. Lactobacillus
1. Pseudomonas spp. d. Propionibacterium (Cutibacterium)
2. Burkholderia e. Eubacterium
3. Stenotrophomonas f. Mobiluncus
4. Shewanella 2. Spore Forming
 Non-Pseudomonadaceae a. Clsotridium
1. Acinetobacter  Gram Negative Anaerobic Cocci
2. Alcaligenes faecalis 1. Veilonella
3. Moraxella lacunata 2. Megasphera
4. Chromobacterium 3. Acidaaminococcus
5. Flavobacteriaceae group  Gram Negative Anaerobic Rods
6. Actinobacillus 1. Bacteroides
2. Fusobacterium
◘ FASTIDIOUS GRAM NEGATIVE BACILLI 3. Leptotrichia
 Neisseria, Moraxella spp. 4. Prevotella
 Sphingomonas paucimobilis (NF), Chryseobacterium (Elizabethkingia) 5. Porphyromonas
 Haemophilus spp.
1. Haemophilus influenza ◘ SPIROCHETES
2. Haemophilus ducreyi  Treponema pallidum
3. Other Haemophilus  Leptospira interrogans icterohemorrhagica
 HACEK  Borrelia
1. Haemophilus aphrophilus 1. Borrelia burgdorferi
2. Aggregatibacter actinomycetemcomitans 2. Borrelia recurrentis
3. Cardiobacterium hominis
4. Eikenella corrodens ◘ ATYPICAL BACTERIA
5. Kingella spp.  Chlamydia (New Taxonomy: Chlamydophila)
 Brucella spp. 1. Chlamydia trachomatis
 Bordetella spp. 2. Chlamydia psitttaci
 Franciscella spp. 3. Chlamydia pneumoniae
 Legionella spp.  Mycoplasma and Ureaplasma
 Pasteurella multicoda 1. Mycoplasma pneumonia
 Gardnerella vaginalis 2. Mycoplasma hominis
 Capnocytophaga spp. 3. Ureaplasma urealyticum
 Calymmatobacterium  Rickettsiaecea
 Streptobacillus moniliformis  Anaplasmaceae
 Spirillum minus  Bartonellaceae
 Coxiellaceae

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 BACTERIAL TAXONOMY AND NOMENCLATURE SPECIES
 Species (abbreviated as sp., singular or spp., plural) is the most basic of the taxonomic groups and can be
Taxonomy is the area of biological science comprising three distinct but highly interrelated disciplines: defined as a collection of bacterial strains that share common physiologic and genetic features and differ
► CLASSIFICATION notably from other microbial species
► NOMENCLATURE
► IDENTIFICATION
Each organism has a scientific label consisting of two part: the genus and species designation
► Genus: the first letter is always capitalized
Classification is a method for organizing microogranisms into groups or taxa based on similar morphologic, ► Species: the first letter is always in lower case
physiologic, and genetic traits. The hierarchical classification system consists of the following taxa designations:
Two components are used simultaneously and are printed in italics or underlined in script.
DOMAIN Bacteria, Archaea, Eukarya
KINGDOM similar Divisions or Phyla (similarities of DNA and RNA) Alternatively, the name may be abbreviated by using the uppercase form of the genus’ firs letter followed by a
DIVISION or PHYLUM similar Classes (equivalent to the Division taxa in botany) period (.)
CLASS similar Orders
The full species name is never abbreviated (e.g., S. pneumonia, S. pyogenes, S. agalactiae, S. bovis)
ORDER similar Families
FAMILY similar Genera
Frequently, an informal designation (e.g., staphylococci, streptococci, enterococci) may be used to label a
GENUS similar Species
particular group of organisms. These designations are not capitalized or italicized.
SPECIES specific Epithet
MICROORGANISMS
“Dear King Philip Came Over For Good Spaghetti”
ACELLULAR CELLULAR
VIRUSES PROKARYOTES EUKARYOTES
NOTE: Tribe is included in the hierarchy of taxonomy (it is in between family and genus) but is not commonly utilized
Eubacteria: true bacteria
in the bacterial taxonomy Fungi
Cyanobacteria: blue-green algae
Protozoan
Archaebacteria: can survive extreme environment
SUBTYPE and SUBSPECIES
BACTERIAL CHARACTERISTICS
SEROTYPE: differentiated based on serologic markers (e.g., E. coli O157:H7)
PROKARYOTIC
BIOTYPE: differentiated based on biochemical tests (e.g., EHEC MUG (-) and SMAC (-))  Pro meaning before and karyo meaning nucleus; organisms existed way before a true nucleus emerged
 Absent nucleus but equipped with nucleoid
 No organelles nor membrane-enclosed structure
 Do not have HISTONES (proteins required for mitotic division) and multiples via BINARY FISSION
Nomenclature is the naming of microorganisms according to established rules and guidelines set forth in the  Have 70s ribosomes
International Code of Nomenclature of Bacteria (ICNB) or the Bacteriological Code (BC).  HAPLOID with a single chromosome
 Unicellular containing both DNA and RNA with an average size of 0.2-5.0 microns
o Smallest living organism (1-3 um): MYCOPLASMA
o Smallest pathogenic bacteria: HAEMOPHILUS DUCREYI
Microbial Identification is the process by which a microorganism’s key features are delineated. Once those features o Largest living organism: BACILLUS
have been established, the profile is compared with those of other previously characterized microorganisms. o Largest pathogenic bacteria: BACILLUS ANTHRACIS
o Longest bacteria (10 um): BORRELIA

FAMILY  Cell wall: with peptidoglycan except Mycoplasma and Ureaplasma

 A family encompasses a group of organisms that may contin multiple genera and consists of organisms with a  Cytoplasmic membrane: Fluid phospholipid bilayer with carbohydrate and sterol
common attribute  Site of energy production: Cytoplasmic membrane
 The name of a family is formed by adding the suffix –aceae to the root name of the type genus  Sire of protein synthesis: Free ribosome
 BIOFILM: the property of bacteria to attach on solid surface
Ex. The Streptococcaceae family type genus is Streptococcus

GENUS
 Genus (Plural: Genera) contains different species that have several important features in common
 Each species within a genus differes sufficiently to maintain its status as an individual species

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 MORPHOLOGIES
COCCI REMEMBER:
Pairs: Diplococci ► BACTERIA: single-cell prokaryotic microorganisms
Oval or Spherical
Chains: Streptococci ► FUNGI: single-cell or multicellular eukaryotic organisms
Clusters: Streptococci ► YEAST: unicellular, eukaryotic organisms
BACILLI ► PARASITE: single-cell or multicellular eukaryotic organisms
Rods Singly, Chains, ► VIRUS: dependent on host cell for survival ∴ NOT considered cellular organisms but rather Infectious Agent
Pallisades (side by side)

Very short Rods COCCOBACILLI RIBOSOMAL UNIT

70s 80s
Tapered end FUSIFORM PROKARYOTES,
Present in EUKARYOTES and all higher organisms
Mitochondrion, and Chloroplast
Club-shaped or Curved VIBRIO 50s 30s 60s 40s
2 rRNAs (23s and 5s) 16s rRNAs 3 rRNAs (28s, 5.8s, 5s) 18s rRNAs
Subunits
complexed with complexed with complxed with complexed with
Spiral or Helical SPIROCHETES 34 proteins 21 proteins 49 proteins 33 proteins
AMINOGLYCOSIDE
CHLORAMPHENICOL
Antibiotic (Autotoxic and
CURVED BACILLI (Toxic to bone marrow)
Nephrotoxic)
Comma-shaped (C-shaped) Vibrio Composition 60% RNA and 40% Proteins 40% RNA and 60% Proteins
S-shaped (Seagull wings) Helicobacte, Campylobacter PRIONS: absent ribosome
Fusiform (Spindle) Shaped Capnocytophaga 16s rRNA genetic sequencing: used to determine bacterial phylogeny
Spiral Spirilum minus, Spirochetes (Borrelia, Leptospira, Treponema)

PROKARYOTE vs. EUKARYOTE

Characteristics Prokaryote Eukaryote
Greek Origin “prenucleus” “true nucleus”
Cell Size 0.20-2.0 um 10-100 um
Plants (Cellulose)
Cell Wall Present (Peptidoglycan) Fungi (Chitin)
Animals and Protozoans (Absent)
Phospholipid bilayer WITHOUT
CHO and sterol component
Cytoplasmic Phospholipid bilayer WITH
(except Mycoplasma and Ureaplasma; they
Membrane CHO and sterol component
contain CHO and sterol in their cell wall to resist
osmotic pressure of the external environment)
ABSENCE of TRUE NUCLEUS with
Nucleus
nuclear membrane or nucleoli nuclear membrane and nucleoli
Genome Location In the nucleoid at the mesosomes In the nucleus
Single, circular chromosomes (some bacteria,
such as Vibrio cholerae, have two chromosomes Multiple linear chromosomes
Chromosomes
and some have linearly arranged chromosome) with histones
without histones
Cell Organelles Absent Present
Ribosomes Present; SMALLER size (70s) Present; LARGER size (80s)
Pili and Fimbriae Present Absent
Site of Energy
Cytoplasmic membrane Mitochondria
Production
Site of Protein
Free Ribosomes Rough Endoplasmic Reticulum (ER)
Synthesis
S: Svedberg unit (unit of sedimentation)

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 PARTS OF A BACTERIAL CELL CAPSULE DEMONSTRATION
SEROLOGIC (ANTIGENIC) STAINING
1. Cell Envelope Serotyping by Quellung (German word meaning “SWELLING”) reaction Negative staining method
1.1. Glycocalyx
1.2. Capsule (aka Slime Layer) ► Neufeld-Quellung Capsullar Antigen: ► HISS staining
1.3. Cell Wall (aka Murein Layer or Peptidoglycan Layer) positive (+) capsular swelling due to Ag-Ab binding ► India Ink (Nigrosin)
1.4. Cell Membrane
2. Pili or Fimbriae NQCA has been replaced by direct detection of capsular antigen using
3. Endospores serological markers (e.g., Latex or Fluorescence)
4. Flagella
5. Inclusion Bodies ► CAPSULAR ANTIGEN:
6. Ribosome K Antigen (Vi Antigen for Salmonella typhi)
7. Cytosol
8. Nucleoid “Nuclear Body” CELL WALL “MUREIN LAYER” “PEPTIDOGLYCAN LAYER”
9. Plasmid
 Defines the shape and strength (resist osmotic pressure) of the bacteria
CELL ENVELOPE  The point of anchorage for flagella, site of attachment, and antibiotic action

 Composed of layers (capsule, cell wall, cell membrane) that surround the bacterium
 NOT ALL organisms have cell wall and capsule but ALL bacteria contain cell membrane  Peptidoglycan (Murein layer) consists of glycan (polysaccharide) chains of alternating
N-acetyl-glucosamine (NAG) and N-acetyl-d-muramic acid (NAM)
GLYCOCALYX o Mycobacterium spp. have an unusual cell wall structure; cell wall contains N-Glycolylmuramic Acid
instead of NAM; has VERY HIGH LIPID CONTENT (creating a hydrophobic permeability barrier)
 Glycocalyx is the OUTERMOST LAYER of the bacteria and is a cell coat (polysaccharide) that can be a:
o Capsule: firmly attached to the cell wall  TRANSPEPTIDASE
o Slime layer: unorganized material, loose – not firmly attached to the cell wall o cross-links the side chains (NAG and NAM) to build the cell wall
o enzyme that is inhibited by penicillin and cephalosporin
accumulation of microorganisms embedded in a polysaccharide matrix (e.g.,
Persister cells: Pseudomonas aeruginosa – PSAE, Staphylococcus auresu –  LYSOZYME (found on mucosal surfaces) kills bacteria by cleaving the glycan backbone of peptidoglycan
STAU, C. albicans)
 Some components responsible for pathogenicity:
65% of nosocomial (hospital-acquired) infections are associated with biofilm  M protein
BIOFILM  Mycolic acid
Can be monomicrobial or polymicrobial
 Gram positive and gram negative cells can lose their cell walls and grow as “L-forms” in media
Quorum sensing: chemical communication within a biofilm supplemented with serum or sugar to prevent osmotic rupture of the cell membrane
► Gram negative: ACYL HOMOSERINE LACTONE (AHL) o Mycoplasma and Ureaplasma are pleomorphic organisms that lack cell wall and only contains
► Gram positive: OLIGOPEPTIDE sterol; making them pleomorphic and sensitive to osmotic pressure

L-forms are organisms that have temporarily lost their cell wall as a result of environmental condition
CAPSULE “SLIME LAYER”
Characteristics GRAM POSITIVE (+) GRAM NEGATIVE (-)
 A firmly attached, well-organized glycocalyx Permeability to Alcohol NO YES
 The slimy area surrounding (external) the cell wall, responsible for mucoid colonies THIN – Single layer
Peptidoglycan THICK - Multilayer
(with outer and inner membrane)
 Demonstration obtained from: Teichoic Acid PRESENT ABSENT
 animal tissue and fluids Lipopolysaccharide
 media containing MILK or SERUM (important in evading host defenses)
Toxin Exotoxin
 Usually made of polysaccharide or polypeptide except D-gultamic acid from B. anthracis Core polysaccharides, Lipid A,
o Polysaccharide: CHO Exotoxin and Endotoxin
o Polypeptide: Amino acid Antigen Somatic (O)
Sugars NAG, NAM
 Prevents phagocytosis and considered a VIRULENCE FACTOR Periplasm and Outer Membrane YES

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 Teichoic acid provide flexibility to the cell wall and binds to Magnesium ions (Mg2+) ENDOSPORE
Lipid A (Endotoxin): essential for identification of true gram negative bacteria, present in the outer membrane
Periplasmic is the space between cell wall and cytoplasm (Periplasmic Flagella aka Endoflagella or Axial filaments)  Target of sterilization
 A resting cell that is highly resistant to dessication, heat, and chemical agents
CELL MEMBRANE  Formed in response to toxic environment

 Surround the cytoplasm for selective permeability (barrier made of phospholipid bilayer)  COMPOSITION: CALCIUM DIPICOLINATE (aka Dipicolinic Acid)
 Site of energy production and metabolism thus considered as the “Mitochondria of Prokaryotic Cell”
 Functions as the golgi apparatus and lysosome transporting nutrients in and out of the cell ETHANOL (HEAT)
used to differentiate Clostridium (+) and Bacteroides (-) by inducing sporulation
SHOCK SPORE TEST
OUTER MEMBRANE
 There are two gram (+) spore-formers, namely CLOSTRIDIUM and BACILLUS
GRAM NEGATIVE OUTER MEMBRANE ► C. tetani: Round, Terminal swollen spores
► C. botulinum: Oval, Subterminal spores
 Contains proteins, phospholipids, and lipopolysaccharide ► B. anthracis: Round, Central spores
 The lipopolysaccharide contains three (3) regions
► O (Somatic) antigen : O-specific polysaccharide CLOSITRIDIUM BACILLUS
► Core polysaccharide Anaerobe and Catalase (-) Aerobic and Catalase (+)
► Lipid A (aka Endotoxin): responsible for producing fever, hypotension, shock, and DIC

PERIPLASMIC SPACE

 Typically found only in gram (-) bacteria; space between the inner and outer membrane
 Consists of gel-like substances that help secure nutrients from the environment
 Also contains several enzymes that:
 degrade macromolecules
 detoxify environmental solutes including antibiotics
AUTOCLAVING is the best way to destroy spores

INNER MEMBRANE
BACTERIAL SPORES FUNGI SPORES
Virulence factor and non-reproductive Reproduction purpose
MESOSOME

FLAGELLA
 Extension of the cytoplasmic membrane appearling like convulated invaginations
 The point of attachment for chromosome, important for DNA replication and cell division
 Organ for locomotion and motility; represents TRUE MOTILITY
 Can be septal (if it occurs at the septum or cross wall) or non-septal (occurring at lateral
o Brownian Motility is due to the movement of molecules surrounding non-motile organisms
mesosomes)
 It contains the protein “FLAGELLIN” which is antigenic and also known as the H antigen
 Motility is best seen at ROOM TEMPERATURE (25oC)
CYTOSOL
o 37oC is INHIBITORY for some bacteria (e.g., Listeria, Yersinia enterocolitica)
 Site of all other biochemical reactions that are not done in the cytoplasmic membrane
Ways to Demonstrate Motility

PILI (Plural: PILUS) or FIMBRIAE

1. FLAGELLAR STAIN (Fisher and Conn, Leifson, Gray): contains TANNIC ACID to precipitate the flagella

 Usually found in gram negative (-) bacterlia (e.g., E. coli, N. gonorrhoeae, and Pseudomonas) 2. HANGING DROP METHOD: observed under OIO
 Hair-like preotinaceous structures made up of the protein “PILIN”

Common “Ordinary” Pili Sex “Conjugative” “Fertility” Pili

For the adherence of For gene transfer (bacterial conjugation)
bacteria to host cell;
1st step in establishing Ex. Escherichia coli
infection
F (Fimbriae) positive Bacteria
3. SEMI-SOLID MEDIA: Sulfide Indole Motility (SIM), Motility Indole Ornithine (MIO) incubated at RT overnight
Ex. Neisseria gonorrhoeae can only conjugate with F (-) bacteria that becomes positive after conjugation
a. Non-motile – growth is limited on the site of inoculation
b. Motile – growth is away from the site of inoculation
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 PATTERN RIBOSOME
INVERTED CHRISTMAS TREE or UMBRELLA Listeria monocytogenes (Motile)
TEST TUBE BRUSH Erysipelothrix rhusiopathiae (Non-motile)  Free ribosomes responsible for protein synthesis; giving the cytoplasm a granular appearance under electron
microscopy
ATRICHOUS MONOTRICHOUS (Polar) LOPHOTRICHOUS ► Bacteria (70s)
► Fungi (80s)
► Virus (acellular)

Flagellum on one side CYTOSOL

No flagellum Tuft of flagella at one end
(e.g., Vibrio)
(e.g., H. pylori)  The fluid portion of the cell; contains no organelles but ribosomes and various types of nutritional storage
AMPHITRICHOUS PERITRICHOUS AMPHILOPHOTRICHOUS granules

NUCLEOID “NUCLEAR BODY”

Flagellum on both ends Flagella all over  The DNA in the bacterial cell is generally confined to this region
Tuft of flagella on both ends
(e.g., Listeria) (e.g. Enterobacteriaceae)  Not surround by a nuclear membrane and does not have mitotic apparatus
PERIPLASMIC FLAGELLA ( aka Axial Filament, Endoflagella)
PLASMID

 A small DNA molecule carrying a resistance gene that confers resistance to antibiotics

NOT a true flagella (not made up with Flagellin) but gives the spirochetes the “corkscrew” motility

TYPES OF MOTILITIES

► Shooting Star and Rapid Darting Motility: Vibrio cholerae

► Tumbling Motility: Listeria monocytogenes (at RT)
► Twitching Motility: Kingella kingae
► Darting Motility: Campylobacter spp. (Other Reference: Vibrio)
► Gliding Motility: Capnocytophaga gingivalis
► Corkscrew Motility: Spirochetes
► Swarming Motility: Proteus spp. (on BAP)

INCLUSION BODIES

 Serves as energy source and food reserves

 These are
► GLYCOGEN
► LIPIDS: Much granules
► POLYPHOSPHATES (for ATP): Babes-Ernst
► PYROPHOSPHATES
► BIPOLAR BODIES

INCLUSION BODIES

► Metachromatic Granules (aka Babes Ernst Granules, Volutin): Corynebacterium diphtheriae

► Halberstaedter Prowazek: Chlamydia trachomatis
► Much Granules: Mycobacterium tuberculosis
► Sulfur Granules: Nocardia and Actinomyces
► Bipolar Bodies: Yersinia pestis

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 BACTERIAL METABOLISM PYRUVIC ACID → ENERGY + BYPRODUCTS
Carried out by obligate aerobes and
Respiration Kreb’s Cycle (Tricarboxylic Acid Cycle)
IRON METABOLISM facultative anaerobes
(Aerobic Process)
Electron Transport Chain Oxygen is the final electron transporter
IRON (Fe3+): in the form of ferric ion, is required for the growth of bacteria because it is an essential component of Release of acid from glucose; efficient energy generating process in which molecular
cytochromes and other enzymes oxygen is the final electron acceptor

Oxidation
SIDEROPHORES (Enterobactin): iron-binding compound produced by E. coli to capture iron by chelating it (leading to Certain anaerobes can carry out anaerobic respiration, in which inorganic forms of
(Aerobic Process)
anemia from chronic infection) oxygen (e.g., nitrate, sulfate) act as the final electron acceptor

CARBOHYDRATE METABOLISM Oxidizers (Non-fermenters) metabolize glucose in the presence of oxygen

Carried out by both obligate anerobes and facultative anaerobes
How bacteria utilize the sugars they ingest:
PRODUCTS: are acids that accumulate in the medium
Carbohydrates → Glucose → Energy (Pyruvic acid) → Energy + byproducts (ATP)  Lactate, Butyrate, Ethanol, Acetoin

GLUCOSE: the starting carbohydrate for bacterial fermentation or oxidation; bacteria metabolizes it into pyruvate by three Fermenters metabolize glucose with or without oxygen
(20 possible metabolic pahtways
CLASSIFICATION ACCORDING TO END PRODUCTS
GLUCOSE → PYRUVIC ACID Alcoholic Fermentation Ethanol (yeast)
PATHWAY NOTES MAJOR ENZYME Homolactic Fermentation Lactic acid only (Streptococcus and Lactobacillus)
Embden-Meyerhof-  Major source (90% glycolysis) yielding 2 ATPs Heterolactic Fermentation Lactic acid, Formic acid, Acetic acid, Alcohol, CO2
Parnas Glycolytic  An ANAEROBIC process occurring in the cytoplasm Pyruvate kinase Propionic acid (Propionibacterium acnes; new name
Propionic Acid Fermentation
Pathway  Carried out by all members of Enterobacteriaceae Cutibacterium acnes)
Fermentation
 10% glycolysis yielding 38 molecules of ATPs (Anaerobic Process)
Lactic acid, Acetic acid, Formic acid, Succinic acid,
Pentose-Phopshate  AEROBIC process
Mixed Acid Fermentation
(Phosphogluconate) Methyl red Test: detects acid production whrn
Glucose-6-
Pathway Glucose → Ribulose-5-phosphate glucose is metabolized (decrease of pH)
phosphate-
or (rearranged into other 3-, 4-, 5-, 6-, and 7- carbon sugars) Acetoin (Acetylmethyl carbinol) and Butanediol
dehydrogenase
Hexose which are neutral end products
(G6PD)
Monophosphate  Used by heterolactic fermenting bacteria (e.g.,
Shunt Lactobacilli and Brucella abortus) which lack some of the Butanediol Fermentation Vogues-Proskauer Test: detects acetoin
enzymes required in the EMP pathway production
 AEROBIC process used by Pseudomonas, Alcaligenes,
Enterococcus facelais, and other bacteria lacking certain Butylene Glycol Pathway: Pyurvate → Acetoin
Entner-Doudoroff glycolytic enzymes Butyric acid, Acetic acid, CO2, Hydrogen (obligate
Butyric Acid Fermentation
Pathway anaerobes)
G6P → Pyruvate and Glyceraldehyde phosphate Organic molecule is the final electron transporter
(funneled into other pathways)

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 PATHOGENECITY and VIRULENCE EXOTOXIN vs. ENDOTOXIN
EXOTOXIN ENDOTOXIN
PATHOGENECITY VIRULENCE ALL major Gram (+) Part of the Gram (-) organisms’ outermembrane
relative ability of a microorganism to cause disease “degree of except P. aeruginosa – gram (-) producing
the ability of a microbe to produce Source Exotoxin A
pathogenicity” usually measured by the numbers of microorganisms
disease in a susceptible individual
(infectious dose, viral load) necessary to cause infection in the host
TOXIN IS PRODUCED BY THE ORGANISM IT IS NOT PRODUCED
Ex. Manner of A metabolic product excreted by living Required cell death for release through
Shigella (Dysentery): infectious dose of 50-100 CFU or 100-200 CFU with scanty, bloody, and painful Release bacteria; does not require cell death cell wall disintegration (lysis)
Enteroinvasive E. coli (Shigella-like infection): infectious dose of 100, 000 CFU with explosive stool Composition PROTEIN (Polypeptide) POLYSACCHARIDE and LIPIDS
Genetics Plasmid or Bacteriophage Bacterial chromosome
BOTH ARE PATHOGENIC, SHIGELLA IS MORE VIRULENT LOCAL: focal, targets specific host cell and SYSTEMIC: all over the body, general and non-
not associated with fever specific associated with:
Effect
(Signs and Cytotoxin: kills host cells  Disruption of clotting, causing clots to form
PATHOGENECITY ISLANDS Symptoms) Enterotoxin: damages GIT cells throughout the body (DIC)
Neurotoxin: interferes nerve impulse  Fever
 Mobile genetic elements that contribute to the change and spread of virulence factors among bacterial transmission  Hypotension, Shock, and Death
populations of a variety of species Example of Tetanus (Lockjaw): neurotoxin
Meningococcemia, Sepsis
Disease Botulism (Floppy Baby Syndrome): neurotoxin
 Evolved from lysogenic bacteriophage and plasmids and are spread
Mode of
Various TNF, IL-1, IL-6
Action
VIRULENCE FACTORS Toxicity HIGH (fatal in microgram quantities or less) LOW (fatal in tens to hundreds of micrograms)
ADHERENCE FACTORS Pili (Fimbriae) Lethal Dose LOW: 1 ug HIGH: hundreds of ug
ANT-PHAGOCYTIC Capsule HEAT-LABILE (Unstable at 60oC)
HEAT-STABLE
FACTORS Self-component of cell wall (M protein, Mycolic acid) except Staphylococcall enterotoxin
Coagulase converts Fibrinogen → Fibrin clot Heat Stability
Can withstand heating at temperatures >60oC for
Fibrinolysin (aka Kinase) dissolves clots Destroyed rapidly by heating at
hours without loss of toxicity
Hyaluronidase or Duran temperatures >60oC
spreading factor  YES; highly immunogenic  NO; poorly antigenic
Reynal Factor Anitgenecity
ENZYMES degrades IgA; allows organism to adhere to mucous  can be converted into toxoid (non-toxic  cannot be converted into toxoid (no
(Anti-toxin
membranes but antigenic) for vaccine vaccine)
Production)
IgA Protease  easily neutralized by anti-toxin  not easily neutralized by anti-toxin
Ex. N. gonorrhoeae, H. influenzae, Limulus Lysate Test
S. pneumoniae
Exotoxin Amoebocyte (WBC) from the horse shoe crab
TOXINS releases a lysate (protein) when endotoxin is
Endotoxin
Leukocidin destroy both neutrophilic leukocytes and present
Test
macrophages
REAGENT: horse shoe crab (Limulus
Ex. Staphylococcus aureus polyphemus) blood
Intracellular survival commonly cause granulomatous lesions
OTHER (+): clumping
Ex. Mycobacterium, Legionella, Brucella,
Listeria, Histoplasma
Yersinia outer-membrane protein inhibit phagocytosis by neutrophils and
(YOPS) macrophages and cytokine production by
macrophages

Page 9 of 40
 BACTERIAL GROWTH REQUIREMENTS OXYGEN CARBON DIOXIDE HYDROGEN NITROGEN
Aerobes 15-21% 0.03%
FASTIDIOUS vs. NON-FASTIDOUS Anaerobes 0% 5-10% 5-10% 80-90%
Microaerophilic 5-10% 8-10% 85%
FASTIDIOUS NON-FASTIDIOUS Capnophilic 15% 5-10%
Complex nutritional and supplemental requirement Basic nutritional requirement
GASPAK JAR CANDLE JAR
FACTORS For Anaerobic Cultivation (5% Co2, 10% H2, 85% N2) For Capnophilic (5-10% CO2) and
Microaerophilic (5% O2) Cultivation
1. CARBON: synthesis of cellular components
2. NITROGEN: synthesis of proteins, RNA, and DNA
3. IONS and MINERALS: Sulfure, Magnesium, Iron
4. SALT
5. WATER and MOISTURE
6. OTHERS
a. PROTEIN: in Egg-based media for MTB (Much granules)
b. X and V Factors: Haemophilius

NUTRITIONAL REQUIREMENTS

PHOTOTROPHIC uses light as energy source INDICATORS Only white, unscented candles should be
Methylene Blue Resazurin used because other types may be toxic to N.
CHEMOTROPHIC uses chemical compounds as energy source; COMMON
AEROBIC Blue Pink gonorrhoeae and N. meningitides
AUTOTROPH (LITHTOTROPH) uses inorganic compounds as carbon source (ex. CO2) ANAEROBIC Colorless or White Colorless
HETEROTROPH (ORGANOTHROPH) uses organic compounds as carbon course (ex. Glucose, Lipid) The candle will burn until the oxygen
CATALYST: Palladium Pellets concentration has been lowered to the point
OXYGEN, CARBON DIOXIDE, NITROGEN REQUIREMENTS → maintains anaerobiasis by removing residual oxygen from the at which it will no longer support the flame.
chamber by combining with H+ to form H2O
Candle jar atmosphere can be achieved by:
OXYGEN 3% (3-5%) CO2
MOST COMMON FAILURE: inactivation of catalyst due to repeated
 organisms requiring oxygen for growth  Pseudomonas use
OBLIGATE (STRICT)  equipped with: superoxide dismutase (SOD)  Neisseria (NP) ► must be replaced or inactivated each time the jar is used — CAPNOHILES: Neisseria and HACEK
AEROBE and catalase that converts toxic products of  Brucella heat the pellets in a dry oven for 2 hours (160-170oC) for
oxygen to non-toxic substances  Francisella rejuvenation
 Clostridium
 organisms not requiring oxygen for growth used as a filter for the remaining filter of the
OBLIGATE (STRICT)  Bacteroides Nitorogen Gas
 dies in the presence of oxygen because they are anaerobic atmosphere
ANAEROBE  Veilonella
not equipped with converting enzymes Silica Gel absorbs water formed whrn H2 combines with
 Actinomyces (Dessicant) free oxygen and catalyst
 organisms (aerobes in nature) that can live with  Enterobacteriaceae H and CO2 facilitate the growth and isolation of anaerobes
FACULTATIVE
our without oxygen  S. aureus
ANAEROBE
 with superoxide dismutase (SOD) and catalase  Streptococcus SALT REQUIREMENTS “HALOPHILIC”
 organisms does not grow well but survives in
 Lactobacillus
AEROTOLERANT the presence of oxygen 6.5% 7.5% 8-10%
 Propionibacterium
ANAEROBE  with superoxide dismutase (SOD) Enterococci Staphylococcus aureus Vibrio except V. mimicus and V. cholera (1-3%)
 C. perfringens
 do not use oxygen in metabolism
 organisms requiring small amount of oxygen  Campylobacter jejuni pH REQUIREMENTS
MICROAEROPHILIC
(5-10% O2, 10% CO2, 85% N2)  Helocibacter influenzae
 organisms able to survive in an unusual condition ACIDOPHILIC NEUTROPHILIC BASOPHILIC (ALKALIPHILE)
EXTREMOPHILE  Bacillus infernus pH 3 pH 6.5-7.5 Ph 8-10
(absence of oxygen and increased temperature)
CARBON DIOXIDE Most bacteria grow well in medium
Requiring an alkaline medium for
 Streptococcus Requiring acid medium for growth with pH between 6.5-7.5
 organisms requiring high concentrations growth
CAPNOPHILIC  Neisseria (P)
(5-10%) of carbon dioxide for optimal growth Ex. L. acidophilus grows on Tomato Commercially prepared media are
 HACEK Ex. Vibrio on TCBS at pH 8.6 (8-10)
SOD and Catalase are protective enzymes used by aerobe that protects them from the toxic effects of oxygen Juice Agar at pH 3.0 buffered in range so that checking
on Alkaline Peptone Water
their pH is rarely necessary

Page 10 of 40
 TEMPERATURE REQUIREMENTS STAGES OF BACTERIAL GROWTH

PSYCHROPHILIC (CRYOPHILIC) MESOPHILIC THERMOPHILIC

<10 oC (10-20oC) 20-40 oC 50-125 oC
”Cold Loving” bacteria “Heat Loving” bacteria

Ex. Bacteria able to grow at moderate Ex.

Listeria in coleslaw food poisoning temperature; best at 30-37oC B. stearothermophilus
and milkborne diseases most of these are pathogenic Sulfolobus
Y. enterolitica in blood bag (Incubator: 35 ± 2oC) Pyrococcus
contamination (indicated by Pyrodictium
presence of bubbles) Thermus aquaticus
Thermoduric: not growing well in the presence at high temperature but can withstand exposure

0.5-45oC Listeria
Campylobacter jejuni
42 oC Acinetobacter baumanii
Pseudomonas aeruginosa LAG PHASE  NO cell division during this stage
65 oC Legionella (hotsprings, airconditioning tower, showerhead) (Rejuvenescence,  Increase (↑) in size but not in number; bacterial enzyme is active
Adjustment,  Growth Rate: 0%
Physiologic)  The stage of adaptation
SMART INCUBATOR

 Mainly for random and faster retrieval when the laboratory professional wishes to access a particular plate  Start of cell division at a constant rate
 Each individual plate has its own shelf LOG PHASE  Organisms are most metabolically active and grows at a maximum rate
o Seprates bacterial plates allowing for optimal temperatures (Exponential)  Growth Rate: increasing
 Culture grows faster in them as compared to traditional incubators  ORGANISMS BECOME SUSCEPTIBLE TO ANTIBIOTICS
o Provides for a homogenous atmosphere and efficient thermal conductivity in the incubator to bring
the culture plate up to optimal conditions faster
 Growth ceases due to the accumulation of waste and toxic product as well as
STATIONARY PHASE depletion of nutrients
most bacteria and viruses 35-37 oC (Plateau) # dying organisms = # viable organisms
Incubation temperature for
most fungi 28-30 oC (Optimum: 37 oC)  Growth Rate: none
aerobes 35-37 oC for 18-24 hours
Incubation temperature and time for
anaerobes 35-37 oC for 24-48 hours
Incubation of cultures for bacterial growth in Diagnostic Laboratories 35 oC # dying organisms > # viable organisms
Pseudomonas and Campylobacter can grow at both 35 oC and 42 oC PHASE OF DECLINE
TEMPERATURE Neisseria meningitidis  Decrease (↓) in the number of viable organisms but not in total bacterial count
Sensitive to changes in
pH Shigella
Most bacteria takes 18-24 hours for growth phase

Page 11 of 40
 BACTERIAL GENETICS Transposable Elements
→ pieces of DNA that move from one genetic element to another
Genetics, the process of heredity and variation, is the starting point from which all other cellular pathways, functions, and → not capable of independent replication
structures originate. The ability of a microorganism to maintain viability, adapt, multiple, and cause disease is determined
by the organism’s genetic composition. Insertion Sequence
genes required for movement among plasmids and chromosomes
(SIMPLE TRANSPOSONS)
The two major classes of nucleic acids are: Composite Sequence genes for movement (highly mobile pieces of DNA) along the chromosome or
1. Deoxyribonucleic acid (DNA) (JUMPING GENES) plasmid as well as other features (drug resstance and toxins)
2. Ribonucleic acid (RNA)
BACTERIAL GENETICS (MUTATION)
NUCLEOTIDE STRUCTURE AND SEQUENCE CONJUGATION
Deoxyribonucleic Acid (DNA) Ribonucleic Acid (RNA)  The sex pilus or transposon (donor) establishes a
Definition conjugative bridge that serves as the channel for DNA
It is a long polymer; has deoxyribose and phosphate It is a short polymer with a ribose and phosphate transfer from donor to recipient cell
backbone having four distinct bases (Thymine, Adenine, backbone with four varying bases (Uracil, Adnine,  Occurs between two living cells, involving cell-to-cell and
Cytosine, Guanine) Cytosine, Guanine) requires mobilization of the donor bacterium’s chromosome
Location  The plasmid (independent circular DNA, non-chromosomal
It is located in the nucleus of a cell and in the element) may be transferred by conjugation, but not all
It is found in the cytoplasm, nucleus, and in the ribosome plasmids are capable of conjugative transfer
mitochondria
Sugar Portion  In addition to chromosomal DNA, genes encoded in
Contains 2-deoxyribose It has ribose extrachromosomal genetic elements (Plasmids, Transporons)
Function may be transferred
RNA is critical for the transmission of the genetic code TRANSDUCTION
Transmission of genetic information acting as a medium  DNA of two (2) bacteria will come together to form other traits
that is necessary for protein creation from the nucleus to
for long-term storage  This process is mediated by viruses that infect bacteria
the ribosome
Predominant Structure (bacteriophage)
DNA is a double-stranded molecule that has a long RNA is a single-stranded molecule which has a shorter o DNA from two bacteria may come together in cone
chain of nucleotides chain of nucleotides cell, thus allowing for recombination
Propagation TRANSFORMATION
RNA does not replicate on its own; it is synthesized from  Involves recipient cell uptake of naked (free) DNA released
DNA replicates on its own into the environment when another bacterial cell (donor)
DNA when required
Nitrogenous Bases and Pairing dies and undergoes lysis
The base pairing is as follows: The base pairing is as follows:  COMPETENT BACTERIA are able to perform transformation
G-C and A-T G-C and A-U (Streptococcus, Haemophilus, Neisseria)

Bacterial chromosome BACTERIOPHAGES: Lysogenic Conversion

→ one (1) single chromosome BACTERIUM PHAGE GENE PRODUCT PHENOTYPE
→ contains all genes essential for viability and exists as a double-stranded, closed, circular, naked macromolecule Vibrio cholerae CTX phage Cholera toxin Cholera
Escherichia coli Lambda phage Shiga-like toxin Hemorrhagic diarrhea
Plasmids Clostridium botulinum Clostridial phage Botulnum toxin Botulism (food poisoning)
→ “miniature” chromosome, double-stranded, closed, circular Corynebacterium diphtheria Corynephage beta Diphtheria toxin Diphtheria
→ do not usually encode for products essential for viability but are inducers Streptococcus pyogenes T12 Erythrogenic toxin Scarlet fever
→ highly transferrable from one bacteria to another
 antibiotic, heavy metal, and ultraviolet resistance BACTERIAL GENE EXCHANGE (RECOMBINATION or HOMOLOGOUS RECOMBINATION)
 pili (fimbriae)
 exotoxins RecA protein: required protein for homologous recombination
 bacteriocins
 nitrogen fixation and degradative enzymes MecA gene: encodes Methicillin resistance; serves as a marker for MRSA identification
 tumors
Once a bacteria attains a foreign DNA from its donor, it will inject the foreign DNA with a homologous recipient,
exchanging the DNA segment

Some segment of the DNA that originated from one bacterial cell (e.g., donor) enters a second bacterial cell (e.g.
recipient) and exchanges with a DNA segment of the recipient’s genome

Page 12 of 40
 METHODS OF MICROBIAL CONTROL
OVERVIEW OF PCR AND DERIVATIONS
MICROORGANISM or INFECTIOUS AGENT
Polymerase Chain Reaction (PCR) is based on three simple (bacteria, virus, fungi, parasite, prion)
steps required for any DNA synthesis reaction: ↓
1. DENATURATION of the template into single RESERVOIR or SOURCE
strands (human, water, air, fomite, insect, body fluid)
2. ANNEALING of primers to each original strand ↓
for new strand synthesis PORT OF EXIT
3. EXTENSION of the new DNA strands from the (secretion, excretion, droplets)
primers ↓
MODE OF TRANSMISSION
(droplet, airborne, contact, vector, vehicle)
↓
PORTAL OF ENTRY
(nose, mouth, mucous membranes, skin, open wound, unsterile equipment)
↓
SUSCEPTIBLE HOST
(patient, elderly, newborn, immunocompromised, healthcare worker)

Ex. Yersinia pestis > Rats (Ratus ratus) > Secretions > Vector: Rat flea (Xenopsylla cheopsis) > Bite wound > Host

SKIN is the primary physical barrier: WARM MOIST SKIN is more susceptible to infection

A person who harbors the etiologic agent but shows no apparent signs or symptoms of
CARRIER
infection or disease
The origin of the etiologic agent or location from which it disseminates (e.g., water,
RESERVOIR
food, insects, animals, other humans)
A living entity (animal, insect, plant) that transmits etiologic agent
required in the pathogen’s life cycle
BIOLOGIC VECTOR
VECTOR Ex. Anopheles minimus var. flavirostris for Plasmodium
not required in the pathogen’s life
MECHANICAL VECTOR
Ex. Periplaneta americanum – household cockroach
A non-living entity that is contaminated with the etiologic agent and as such is the mde of
transmission for that agent
VEHICLE
VEHICLE: air, water, soil
FOMITE: door knobs, curtains, table tops
A larger than normal number of diseased or infected individuals that occurs over a
relatively short period
OUTBREAK

An occurrence of a disease in a location where it is not normally found

EPIDEMIC A larger than normal number of diseased or infected individuals in a particular location
ENDEMIC A disease constantly present in a geographic location
PANDEMIC An epidemic affecting multiple continents
Laboratory-based characterization of etiologic agents designed to establish their
relatedness to one another during a particular outbreak or epidemic
STRAIN TYPING
PHAFE TYPING: bacteria
PULSED FIELD GEL ELECTROPHORESIS: Gold standard
SIGNS Measurable indications or physical observations
SYMPTOMS Indicators described by the patient
An asymptomatic period and the time from acquiring infection to having the first signs and
INCUBATION PERIOD
symptoms
Page 13 of 40
 TRANSMISSION-BASED PRECAUTION

STANDARD PRECAUTION  Measles, Varicella, Tuberculosis, Smallpox

AIRBORNE
 For possible inhalation of droplets (≤ 5 um) that can travel more than 3 feet
Treat all patients and body fluids (except sweat, done on babies with cystic fibrosis with excessive salt in their swear)  Healthcare worker wears N95 mask or HEPA respirator (snug fit) while the patient wears a surgical
as potentially infectious and hazardous mask
 Private or solo room that has monitored negative air pressure, 6-12 air change/hour
► Perform hand hygiene before and after every patient contact
 Invasive Haemophilus influenzae Tyoe B infection including Meningitis, Pneumonia, Epiglottis, Sepsis
► Clean and reprocess shared patient equipment
 Invasive Neisseria meningitidis infection including Meningitis, Pneumonia, Sepsis
► Use personal protective equipment (PPE) when risk of body fluid exposure
 Diphtheria (Pharyngeal)
► Follow respiratory hygiene and cough etiquette

DROPLET
 Mycoplasma pneumonia
► Use and dispose of sharps safely
 Streptococcal Pharyngitis, Pneumonia, or Scarlet fever in infants and young children
► Use aseptic technique
 Adenovirus, Influenza virus, Mumps, Parvovirus B19, Rubella
► Perform routine environmental cleaning
 For possible inhalation of droplets (>5 um) that cannot travel more than 3 feet
► Handle and dispose of waste and used linen safety
 Healthcare worker wears surgical mask (better with face shield) when working within 3 feet of patient
 Private room or cohort of 3 patients with similar conditions
 Gastrointestinal, respiratory, skin, or wound infections, or colonization with multidrug-resistant bacteria
EXPOSURE CONTROL PLAN Clostridium difficile
 For diapered or incontinent patients: Escherichia coli O157:H7, Shigella, hepatitis A virus, or rotavirus
 Identifies tasks that are hazardous to employees and promotes employee safety  Respiratory syncytial virus, parainfluenza virus, and enterovirus infections in infants and young children
 This is the responsibility of the microbiology supervisor and laboratory director  Skin infections such as diphtheria (cutaneous), herpes simplex virus (neonatal or mucocutaneous),
 Post-exposure investigation is done by the microbiology supervisor and safety officer impetigo, major abscesses, cellulitis, or decubiti, pediculosis (lice infestation), scabies (mite infestation),

CONTACT
staphylococci furunculosis (boils) in infants and young children, zoster (disseminated or in the
immunocompromised host)
 Viral hemorrhagic infections (Ebola, Lassa, or Marburg)
FVE (5) MOST COMMON LABORATORY-ACQUIRED INFECTIONS (Not in Order)  Place patient in private room without special air handling or ventilation or cohort patients
 HCWs should:
Shigellosis  wear gloves, mask, and eye protection (activities that are likely to generate body fluid splashes)
FECAL-ORAL  wash hands with a special antimicrobial agent or a waterless antiseptic agent
Salmonellosis
Tuberculosis  wear a spill-proof gown during procedures likely to generate splashes
AIRBORNE  ensure reusable equipment is properly sterilized and single-use items are properly discarded
Brucellosis
Hepatitis NEEDLESTICK

Males and younger employees (17-24 years old) are involved in laboratory-acquired infections than females and older PERSONAL PROTECTIVE EQUIPMENT (PPE)
employees (45-64 years old)
► plastic shiels or goggles to protect workers from droplets
► disposal containers ofr sharp objects
► holers for glass bottles
► trays to carry smaller hazardours items (e.g., blood culture bottle)
HANDWASHING
► handheld pipetting devices
► impervious gowns, laboratory coats, disposable gloves, masks
THE MOST IMPORTANT PRACTICE TO PREVENT THE SPREAD OF DISEASE IS HANDWASHING
► safery carriers for centrifuges (especially those used in the Acid-Fast Bacteriology laboratory)
(entire duration is 40-60 seconds)
► HEPA respirators
1. Wet hands with warm water
DONNING DOFFING
2. Apply germicidal or anti-microbial soap
3. Rub to form a lather, create friction, and loosen debris 1. Wash or sanitize hand 1. Gloves
4. Thoroughly clean between fingers, including thumbs, under fingernails and rings, and up to the wrist, for at least 2. Gown 2. Gown
15-20 seconds. 3. Mask 3. Goggles
5. Rinse hands (one at a time) in a downward position 4. Goggles 4. Mask
6. Dry with a paper towel 5. Gloves 5. Hand washing
7. Turn off faucets with a clean paper towel to prevent recontamination
Minimum required PPE: GLOVES
VISIBLY SOILED HAND: Handwashing Minimum required vaccination: Tetanus, Dephtheria, Acellular Pertussis (TDAP), Hepa B, Influenza
NOT VISIBLY SOILD HAND: Hand sanitizing with 70% alcohol Healthcare worker required vaccination: Minimum + Varicella, Measles, Mumps, Rubella

Page 14 of 40
 BIOLOGICAL SAFETY CABINET (BSC)
BIOLOGICAL SAFETY LEVEL (BSL) CLASS I Open-fronted negative pressure, ventilated cabinets
 Contaminants; no known potential of infection healthy  Bacillus subtilis Sterilize both the air entering and circulating the cabinet and exhaust air
CLASS II
BSL-1 people  Mycobacterium gordonae Filters the air that flows over the infectious material as well as air to be exhauster
“Laminar Flow BSC”
 Poses MINIMAL threat to laboratory workers  Naegleri gruberi Most common in laboratory; with VERTICAL LAMINAR FLOW
 Bacillus anthracis CLASS III Provides highest level of safety; all air entering and leaving the cabinet is sterilized
 Microorganisms associated with human diseases that are
 Yersinia pestis
rarely serious and for which preventive or therapeutic
BSL-2  Staphylococcus BIOLOGICAL SAFETY CABINET (BSC)
interventions are often available
 Enteric pathogen Allow room (unsterilized) air to pass into the cabinet and around the area and material within,
 May pose MODERATE threat to laboratory workers CLASS I
 HIV and HBV sterilizing only the air to be exhausted
 Mycobacterium tuberculosis A Most hospital clinical microbiology laboratory scientists use Class II A cabinets
 Rickettsiae 70% recirculated, 30% exhausted and self-contained; exhaust and circulating air is
 Those not normally encountered in a clinical laboratory and  Francisella tularensis IIA 1
CLASS II sterilized
BSL-3 with potential for aerosol transmission (inhalation)  Brucella Similar to II A1 but has 100 lfm intake air velocity and plenums are under negative pressure
 May pose HIGH risk to laboratory workes  Coxiella (MR, FBC) IIA 2
to room; exhaust air can be ducted to the outside through a canopy unit
 Coccidiodes immitis It is selected if radioisotopes, toxic chemicals, or carcinogens will be used; air is discharged
 Arbovirus B
outside the building
 MERS-COV CLASS II
IIB 1 30% recirculated, 70% exhausted
 Poses EXTREME RISK to laboratory workers  EBOLA IIB 2 No recirculation
 Dangerous and exotic agents causing life threatening  Folivirus
BSL-4 Air coming into and going out of the cabinet is filter sterilized and the infectious material
diseases for which effective treatments are limited  Arenavirus CLASS III
within is handled with rubber gloves that are attached and sealed to the cabinet
 These organisms often encountered in a research institution  Arbovirus
CLASS IIA+: for COVID19
 Small pox virus
BIOLOGICAL SAFETY CABINET (BSC)
BSL-1 BSL-2 BSL-3 BSL-4
APPLICATIONS
 Controlled access  Controlled access  Air tight when  Air tight
BSC FACE Non-Volatile Toxic Volatile Toxic
 Hand washing sink  Hand washing sink disinfecting  Self-closing, double- AIRFLOW PATTERN
CLASS VELOCITY Chemicals and Chemical and
 Sharp hazards  Sharp hazards  Self-closing, double- door access
Radionuclides Radionuclides
warning policy warning policy door access  Controlled access
In at front through HEPA to the outside or When exhausted
 Personal protective  Physical  Controlled access  Sharp hazards I 75 Yes
into the room through HEPA outdoors
equipment containment device  Personal shower out warning policy
70% recirculated to the cabinet work area
 Laboratory bench  Personal protective (risk-based  Hand washing sink
through HEPA; 30% balance can be Yes
 Autoclave equipment enhancement)  Sealed penetrations IIA 1 75 No
exhausted through HEPA back into the (minute amount)
 Laboratory bench  Sharp hazards  Physical
room or to outside through a canopy unit
 Autoclave warning policy containment device
Similar to II A1 but has 100 lfm intake air When exhausted
 Hand washing sink  Positive pressure
 Sealed penetrations protective suit velocity and plenums are under negative outdoors
IIA 2 100 Yes
II pressure to room; exhaust air can be ducted (formally ‘B3”)
 Physical  Laboratory bench
containment device  Autoclave to the outside through a canopy unit (minute amount)
 Powered air  Chemical shower out 30% recirculated , 70% exhausted;
Yes
purifying respirator  Personal shower out IIB 1 100 Exhaust cabinet air must pass through Yes
(minute amount)
(risk-based  Supply and exhaust dedicated duct outside through HEPA filter
enhancement) HEPA filter No recirculation; total exhaust to the Yes
IIB 2 100 Yes
 Laboratory bench  Effluent outside through a HEPA filter (small amounts)
Autoclave decontamination
Exhaust HEPA filter system
(risk-based RECALL
enhancement)
 Effluent ► Sealed bucket centrifuge containing liquid media should be unloaded in BIOSAFETY CABINET; wait for 15
decontamination miutes for the aerosol particle to settle
system (risk-based
enhancement)

Page 15 of 40
 METHODS OF MICROBIAL CONTROL STERILIZATION (BIOCIDE)

STERILIZATION Sterilization is the complete destruction and removal of all forms of microbial life (pathogens and non-pathogens,
Physical Method Chemical Method vegetative cells) including spores
◘ Moist Heat ◘ Ethylene oxide (Gas Sterilant)
 Autoclave ◘ Formaldehyde vapor and Vapor phase H2O2 PHYSICAL METHOD OF STERILIZATION
 Tyndallization ◘ 2% Glutaraldehyde
 Inspissation ◘ Peracetic acid 1. Moist Heat
◘ Dry Heat
 Oven  Destroy microorganism by:
 Incineration ► coagulation of enzyme and structural proteins
 Direct Flaming ► degradation of nucleic acid
 Cremation  The fastest and simplest method of sterilization
◘ Filtration
◘ Ionizing Radiation (Cold Sterilization) AUTOCLAVE TYNDALLIZATION INSPISSATION
◘ Other Principle: Stream under pressure Discontinuous / Fractional / Intermittent Methods
 Low (Cold Temperature)
 Osmotic Pressure *pressure is used to raise temperature not Day1: Alternate heating (kills vegetative cells)
 Dessication to kill the organisms Day 2: Incubation (spores germinate)
 Lyophilization Day 3: Heating (kills remaining vegetative cells and spores)
Biological Indicator: Principle: Flowing Steam using Principle: Thickening
DISINFECTION Bacillus (Geobacillus) stearothermophilus Arnold Sterilizer through Evaporation
Physical Method Chemical Method spores incubated at 56oC (done every
◘ Pasteurization ◘ Antiseptics week) 100oC for 30 minutes for 3 75-80oC for 2 hours for 3
◘ Boiling  Alcohol consecutive days: sterilizes consecutive days:
◘ Ultraviolet  Iodophor  acts by coagulating or denaturing media containing milk or serum sterilizes egg-based and
 Chlorhexidine enzymes and proteins high protein media
 Hexachlorophene Day 1: destroys vegetative cells
 10% Hydrogen peroxide  121oC (250F) for 60 minutes for Day 2: destroys spores Ex. Lowenstein-Jensen
◘ Disinfectant PRIONS (infectious proteins) Day 3: destroys remaining spores medium for MTB
 Halogens
 Heavy metals  121oC, 15 pounds pressure per
 Aldehydes square inch (psi), 15-30 minutes:
 Quaternary Ammonium Compound (QUATS) used and unused media
 Phenolics
 121oC (250F) for 15 minutes Most
effective method of sterilization of
culture media and surgical
instruments

 Infectious medical waste is often

sterilized at 132oC (270oF) for 30-60
minutes to allow penetration of the
steam throughout the waste and the
displacement of air trapped inside
the autoclave bag

Page 16 of 40
2. Dry Heat 4. Ionizing Radation (Cold Filtration)

 It kills microorganism by denaturation of proteins (oxidation); “sterilization without water”  Exposure to HIGH ENERGY GAMMA rays (uses short wavelength but high energy gamma rays); for
 It is utilized for the sterilization of glasswares, metals, oil products, petrolatum, and powders disposable syringe, gloves, and catheters
 Causes DNA mutation and peroxidase generation
OVEN INCINERATION  Also used to pasteurize meat products
Biological Indicator: Bacillus subtilis  870-980oC; hazardous material  Indicator: Bacillus pumilus
var niger spores incubated at 35-37oC  300-400oC burning to ashes
 Most common method of treating infectious waste
 160-180oC for 1.5-3 hours  Safest method to ensure that no infective materials LOW (COLD) TEMPERATURE OSMOTIC PRESSURE
 Sterilizes glassware, metal remain in samples or containers when disposed
instruments, oils, and certain fatty o Prions, infective proteins, are not eliminated during ► Bacteriostatic; cold temperature reduces the ► Use of high concentrations of sugars and salts
acid substances which are not conventional methods ∴ incineration is recommended rate of metabolism (plasmolysis)
permeable to water (i.e. oil,  Disadvantage: production of toxic emission and presence of ► Important in food microbiology ► Bacteriostatic; cells may not die but usualy stops
petrolatum, or powders) heavy metals in ash (RA 8749 - Philippine Clean Air Act) ► 2-8oC for 73 hours kills agents of syphilis growing as water leaves the cell
DIRECT FLAMING CREMATION
 For inoculating loops and needles
to avoid over-inoculation and  To control diseases
ensure individual colonies DESSICATION LYOPHILIZATION

► Bacteriostatic; destroys bacteria through ► Destroys bacteria through changes in proteins which
BACTERIA THAT REMAINS ACTIVE IN DRY ENVIRONMENT metabolism disruption by removing water decreases in chemical reactions

► Neisseria gonorrhoeae: viable for 1 hour

► Mycobacterium tuberculosis: viable for several months CHEMICAL METHOD OF STERILIZATION
► Bacillus and Clostridium: viable for 10 years
Indicator: Bacillus subtilis var. globijii

3. Filtration  most commonly used chemical sterilant

Ethylene oxide (Gas Sterilant)  used in gaseous form of sterilizing heat sensitive objects disinfectant
 Sterilize heat-sensitive materials; antibiotic solutions, toxic chemicals, vaccine, carbohydrates, radioisotopes “COLD STERILIZATION” for machine that cannot be autoclaved or heated
o For urea broth and sugar fermentation broth  have strict requirements for concentration and humidity
 explosive; must be mized with nitrogen or carbon dioxde before use
LIQUID FILTRATION AIR FILTRATION  450-700 mg/L for 55-60oC for 2 hours
Pulling the solution through a Cellulose Acetate Using High Efficiency Particulate Air (HEPA) filters Formaldehyde vapor and
 used to sterilize HEPA filters in BSCs
or Cellulose Nitrate Membrane with a vacuum designed to remove organisms larger than 0.3 um Vapor phase of Hydrogen peroxide
(0.22 um) (bacteria, fungi) from isolation rooms, operating rooms,  SPORICIDAL; kills spores in 3-10 hours
2% Glutaraldehyde
and biologc safety cabinets  used for medical equipment (ieg.g, bronchoscope); it does not
“COLD STERILIZATION”
corrode lenses, metal, or rubber
CRITICAL STERILIZING: eradicate vegetative 0.45um: for removal of more bacteria and fungi Paracetic acid  effective in the presence of organic material and also used for the
cells and ideal for parenteral solutions and “COLD STERILIZATION” surface sterilization of surgical instruments
alcohol

Viruses (0.01-0.3 um) can pass through these filters due to thei relatively small size RECALLS
“Filterable Agents of Infection”
► Sterilization of culture media: AUTOCLAVE or MEMBRANE FILTRATION
Consists of multiple layers of fibrous or granular material ► Sterilization of Evacuated tubes: GAMMA RADIATION; NOTautoclave and ethylene oxide gas (due to changes
in pressure)
DEPTH FILTER 1. Berkeflied filter (Diatomoceous) ► Sealed bucket centrifuge containing liquid media should be unloaded in BIOSAFETY CABINET; wait for 15
2. Asbestos (Seitz): 98% effective miutes for the aerosol particle to settle
3. Chamberlain filter (Unglazed porcelain)
1. Millipore (0.22 um): 100% bacterial sterility
MEMBRANE FILTER
2. Cellulose Acetate or Cellulose Nitrate Membrane (0.22 um): liquid filter

Page 17 of 40
DISINFECTION CHEMICAL METHOD OF DISINFECTION

Disinfection refers to the destruction and removal of pathogen but not necessarily all microorganisms and their spores Antiseptic

PHYSICAL METHOD OF DISINFECTION Antiseptic is a chemical germicide for use on the skin or tissue and not ot be substituted for a disinfectant

 often used to destroy pathogen (ie. Salmonella, Streptococcus, Brucella, Listeria, M. bovis) 1. Alcohols (Non-sporicidal): 70% ethanol or Isopropyl alcohol
in milk (Grade A – safe for consumption) and beverages  75% ethanol is more effective than 95% ethanol since water is necessary for protein denaturation (makes
 used to lower total bacterial count by 95-99% however, this method cannot eliminate lipid soluble); hydrolyzing bonds in protein molecules make the killing of microorganisms more effective
bacterial endospores  Ethyl or Isopropyl: non-sporicidal and evaporates quickly
 PHOSPHATASE: should be negative (-) to test for the success of pasteurization  Other Alcohols: Benzyl Alcohol
Pasteurization Ideal for milk products; can
Ultra High Temperature (UHT) 140oC for 3 seconds 2. Iodophors: Iodine + Detergent or Neutral Polymer (e.g., Povidone)
be stored at RT for months
Flash Pasterurization or Quick heating and Tincture of Iodine: Idone in Alcohol
72oC for 15 seconds  BEST ANTISEPTIC AGENT: Iodine Tincture (70% Alcohol + 2% Iodine)
High Temperature Short time (HTST) immediate cooling
Batch Method or Destroys milk-brone o Iodine is usually prepared as a tincture with alcohol
63oC for 30 minutes  70% ethanol, followed by an iodophor is the most common compound used for skin disinfection before
Low Temperature Holding (LTH) pathogens; reduces spoilage
 Kills all vegetative organisms but not all spores or viruses; 100oC for 15 minutes drawing blood for cultures or before surgery
Boiling
 For surgical instruments
3. Chlorhexidine
 Non-ionizing radiation using UV light (long wavelength)
 Uses MERCURY ARC LAMPS with a wavelength of 240-280 um (LethaL 260 um)
4. Hexachlorophene
Ultraviolet  Damages cellular DNA by producing thymine dimers and water microorganisms are
(< 400 nm) destroyed
5. 10% Hydrogen peroxide
 Do not penetrate well and organism must have direct surface exposure (e.g., working
 Often used for cleansing wounds
surface of BSC) to be effective
UV (CC): very short wavelength with respect to Visible (400-700 nm) and Infrared light (>700 nm)
Disinfectant
UV (Micro): long wavelength with respect to Gamma rays

Disinfectant is applied to non-living surfaces such as countertops and handrails

VINEGAR (Acetic Acid) is a natural disinfectant
GRADE A MILK
1. Halogens (Chlorine, Iodine, Fluorine)
Obtained only from healthy cows with bacterial content <75,0000 bacteria/mL when raw and will not exceed 15,000
 Halogens, especially Iodine and Chlorine are frequently used as disinfectant
bacteria/mL once pasteruized
 Halozone (parasulfone dichloraminobenzoic acid): disinfect drnking water
BEFORE PASTEURIZATION: ≥ 75,000 CFU/mL
IODINE CHLORINE
AFTER PASTEURIZATION: < 15,000 CFU/mL
prepared either as a tincture with most often used in the form and the active component of 5.25%
Standard Plate Count: Estimate of the total number of viable aerobic bacteria present in raw milk alcohol or as an iodophore Sodium Hypochlorite (NaOCl) aka Household Bleach
coupled to a neutral polymer
(e.g., povidone-iodine) Recommended dilution 1:10
Porous Surface 1:10
must remain in contact with the Smooth, Hard Surface 1:100
Large Spill 1:5
skin for 60 minutes
Routine 1 minute
HBV 10 minutes
Inactivates
HIV 2 minutes

SODIUM THIOSULFATE: neutralizes chlorine

Page 18 of 40
 CHLORINE-RESISTANT ORGANISMS SUMMARY OF INDICATORS

► Parasites: Acanthamoeba, Toxoplasma, Cryptosporidium, E. histolytica, G. lamblia MOIST HEAT (Autoclave): Geobacillus (Old Name: Bacillus) stearothermophilus spores at 56oC
► Bacteria: Chyrseobacterium (Elizabrthkingia), Methylobacterium, Empedobacter, Sphingobacterium, DRY HEAT (Oven): Bacillus subtilis var. niger (Bacillus atrophaeus) at 35-37 o C
Aeromonas, Ionizing Radation: Bacillus pumilus
Ethylene oxide: Bacillus subtilis var. globiji
ACID-RESISTANT: Shigella, Enteroviruses Membrane Filter: Pseudomonas diminuta
ACID-SENSITIVE: Coliforms, Rhinovirus

Shigella is ACID-SENSITIVE in specimen, in-vitro but ACID-RESISTANT in the GIT

THERMAL DEATH POINT (TDP): lowest temperature at which a bacterial suspension is killed in 10 minutes

2. Heavy Metals (Hg, Cu, Ag, Mn) THERMAL DEATH TIME (TDT): shortest period needed to kill a bacteria under specific temperature and condition
 No longer recommended; destroys by inactivating and precipitating cell proteins
DECMAL REDUCTION TIME (DRT or D/D Value): time (minutes) to reduce bacterial spores or population by 90% at
Active component of merthiolate (sodium ethyl merucrithiosaliylate); toxic to a specifiec temperature; widely used in the food industry
Mercury
humans and the environment
Copper An algaecide
Used in the form of 1% AgNO3 in CREDE’S PROPHYLAXIS for infants with eye
Silver infection known as opthalmia neonatorium caused by N. gonorrhoeae (now using
Erythromycin)
Manganese

3. Aldehydes (1-8% Formaldehyde or Glutaraldehyde)

 TOXIC TO HUMANS; not used as surface disinfectants due to their irritating fumes
 Antibacterial Effect: inactivation of proteins, nucleic acids, kills endospores

4. Quaternary Ammonium Compounds “QUATS” (Benzalkonium Chloride or Zephiran)

 Low toxicity, used to disinfect bench tops or other surfaces in the laboratory
 ORGANIC SUBSTANCE: rapid inactivation (should be absorbed first prior to application of disinfectant)
 P. auruginosa grown in ammonium acetate media is RESISTANT to QUATS
 Disadvantages: Non-sporicidal, Non-tuberculoidal

5. Phenol (Tuberculoidal)
 STANDARD DISINFECTANT in the laboratory in which other chemical disinfectant are compared
 Phenol Coefficient: expression of bacterial powder of particular substance as compared to pure phenol

ℎ𝑖𝑔ℎ𝑒𝑠𝑡 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑜𝑓 𝑑𝑖𝑠𝑖𝑛𝑓𝑒𝑐𝑡𝑎𝑛𝑡 𝑡ℎ𝑎𝑡 𝑐𝑎𝑛 𝑘𝑖𝑙𝑙 𝑜𝑟𝑔𝑎𝑛𝑖𝑠𝑚𝑠 𝑎𝑡 𝑎 𝑔𝑖𝑣𝑒𝑛 𝑡𝑖𝑚𝑒

𝑷𝒉𝒆𝒏𝒐𝒍 𝑪𝒐𝒆𝒇𝒇𝒊𝒄𝒊𝒆𝒏𝒕 =
ℎ𝑖𝑔ℎ𝑒𝑠𝑡 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑜 𝑓 𝑝𝑢𝑟𝑒 𝑝ℎ𝑒𝑛𝑜𝑙 𝑡ℎ𝑎𝑡 𝑐𝑎𝑛 𝑘𝑖𝑙𝑙 𝑜𝑟𝑔𝑎𝑛𝑖𝑠𝑚𝑠 𝑎𝑡 𝑎 𝑔𝑖𝑣𝑒𝑛 𝑡𝑖𝑚𝑒
Gram (+): S. aureus
Gram (-): S. typhi

PC > 1 Disinfectant is better than phenol

PC < 2 Disinfectant is less than phenol; needs improvement
PC = 1 Same efficiency

 Antibacterial Effect: disrupts cell membrane resulting in leakage of cell contents

HIGH CONCENTRATIONS LOW CONCENTRATIONS

Used as disinfectant Used in soaps

 ENDPOINT: lowest concentration that kills test organisms in 10 minutes at 20oC

Page 19 of 40
 QUALITY CONTROL IN MICROBIOLOGY LABORATORY AUTOCLAVE Spore test weekly
BALANCES Check for accuracy annually
QUALITY ASSESSMENT A program that monitors the total testing process with the aim for providing the BSCs Check airflow annually
Former: Quality Assurance highest quality patient care CENTRIFUGE Check speed (RPM) every 6 months
A system that verifies the reliability of analytical test results through the use of CO2 INCUBATOR Check temperature and CO2 daily
QUALITY CONTROL
standards, controls, and statistical analysis GAS PAK Check for anaerobiosis with Methylene blue strip
A comprehensive program in which all areas of operation are monitored to ensure GRAM STAIN Control slides (E. coli, S. aureus) weekly
QUALITY SYSTEM
quality with the aim of providing the highest quality patient care TEMPERATURE Daily checks for incubators, water baths, refrigerators, and freezers
MICROSCOPES Clean and adjust daily
 Thermometers should be checked periodically against a reference thermometer from the
National Institute of Standards and Technology (NIST) STRAINS USED IN QUALITY CONTROL OF ROUTINE ANTIMICROBIAL SUSCEPTIBILITY TESTS
 Thermometers that differ by more than (>) 1oC from the reference thermometers should Bacteroides fragilis ATCC 25285
Anaerobes
be disposed Eubacterium lentum ATCC 43055
Thermometer o REFERENCE CALIBRATION POINT (oC): 0, 37, 56 Enterococcus faecalis ATCC 51299 Synergy screen test (positive)
Calibration o Other: -20 oC and 2-8 oC Monitors thymidine and synergy test for
Enterococcus faecalis ATCC 29212
o Temperature Factor: +0.1 Enterococci and S. aureus (negative)
 Checked daily for the presence of gas bubbles to ensure accuracy of reading Escherichia coli ATCC 25922 Gram-negative (-) bacteria
 Thermometers are easier to read if they are permanently immerse in glycerol since it prevents Escherichia coli ATCC 35218 Gram negative (-) bacteria β-lactamase positive (+)
temperature fluctuations when the door is opened Haemophilus influenza ATCC 49247 H. influenza (non-β-lactamase)
Carbon Extended spectrum β-lactamases or ESBL
Dioxide  The percentage of carbon dioxide must be check daily Klebsiella pneumonia ATCC 700603
(screening and confirmatory tests)
Incubator Neisseria gonorrhoeae ATCC 49226 N. gonorrhoeae β-lactamase negative (-)
Centrifuge  The speed or revolution per minute (rom) must be checked twice a year using a tachometer Monitoring of the susceptibility media
Pseudomonas aeruginosa ATCC 27853
 All media should be checked based on their performance and sterility, and records should be (calcium and magnesium content)
kept for at least two years Staphylococcus aureus ATCC 25923 Gram-positive (+) bacteria
 The criteria for culture media quality control are established by the Clinical and Laboratory Staphylococcus aureus ATCC 29213 Gram-positive (+) bacteria (MIC)
Culture Standards Institute (CLSI) Staphylococcus aureus ATCC 43300 Oxacillin-resitant
Media  The organisms that are selected for the quality control should represent the most fastidious Streptococcus pneumonia ATCC 49619 Streptococcus pneumonia and other streptococci
organisms
 Media that did not undergo quality control must still undergo observation for moisture, sterility, MICROSCOPY
breakage, and appearance
 Reagents should be tested daily with both positive (+) and negative (-) controls
Reagents MAJOR TYPES OF MICROSCOPES
 Bacteriologic stains should be checked at least daily, or if not possible, once a week
 The most common microscope for gram staining (GS), acid fast staining (AFS), and
 The recommended control organisms are specific strains from the American Type Culure potassium hydroxide (KOH)
Collection (ATCC)
Objective Lens Ocular Lens Total Magnification
 Susceptibility testing of control organisms is usually done for 20-30 days Color
(Initial) (Further) (Ocular x Objective)
Antimicrobial o All results from the 20-to-30-day evaluation should be kept while the antimicrobial BRIGHTFIELD
Scanner 4x RED 40x
Susceptibility agent is being used MICROSCOPE
Low Power 10x YELLOW 100x
 Antimicrobial solutions must not be frozen after thawing 10x
High Power 40x BLUE 400x
 150-200 isolates with comparable growth rates and different levels of susceptibility to the
Oil Immersion 100x WHITE 1000x
agent are tested by both the standard disk diffusion and dilution MIC tests
Used for stained and unstained samples
 All tests performed on patients must be subjected to proficiency testing twice a year
Personnel DARKFIELD  Used for fluorescent stains and to spot the motility of spirochetes
 Participation in continuing education is another form of quality control
Competence MICROSCOPE  Has higher resolving power than brightfield
PHASE  For inclusion bodies (seen on virus and chlamydia), living cells
 Should be grown in a large volume of broth and divided into vials to last for a year
Stock Culture CONTRAST  MICROLYMPHOCYTOTOXICITY TEST for HLA
 To maintain the viability of an organism, it should be subcultured twice after thawing
MICROSCOPE  Good for KOH mount
 Requires fluorescent stain (Calcofluor white, Acridine orange)
FLUORESCENT
► Daily monitoring of the temperature is required for incubator, anaerobic chamber, water bath, heating blocks,  UVL provided by mercury arc lamp
MICROSCOPE
refrigerator, and biofreezer  Substitute: Dark Field Microscopy
► For microscopes, its functionality should be checked quarterly while centrifuge and biohazard hoods should be ELECTRON  Utilized for viral morphology
checked biannually and at least once a year, respectively MICROSCOPE  RESOLUTION: 0.5 nm

Page 20 of 40
 SPECIMEN, COLLECTION, TRANSPORT, AND PROCESSING TRANSPORT AND HOLDING MEDIA

 Collection of specimen for culture must be done within 72 hours (acute phase) of illness or within 2-3 days for Equipped with nutrients (special preservatives) to maintain growth, buffer to maintain pH, and a small amount of agar
viral infections in accordance with ASEPTIC TECHNIQUE observation to maintain moisture when delayed for >2 hours.
 collection containers (sterile, leak-proof, non-breakable) and must be properly labelled (including the specific
anatomic site or source of specimen, antibiotics provided if possible) Many organisms are susceptible to environmental conditions such as:
► Presence of Oxygen: anaerobic bacteria
 Adequate amount must be collected and must be transported without delay – within 30 minutes to 2 hours ► Changes in temperature: Neisseria meningitidis
o Viability Ensurance during Delay: use of special preservatives or holding media for transportation of ► Changes in pH: Shigella
specimens delayed for > 2 hours

PRIORITIZATION OF SPECIMENS HOLDING MEDIA: designed to preserve the viability of microorganism in the specimen but do not allow multiplication
1 Critical / Invasive CSF, Amniotic fluid, Blood, Pericardial fluid, Heart valves
2 Unpreserved Feces, Sputum, Wound drainage Stuart broth Multi-purpose Transport Media bacteria and viruses
3 Quantitation required Catheter tip, Urine, Tissues for quantitation
4 Preserved Urine, Feces, Swab in holding media Amie’s or Stuart’s medium Respiratory samples
5 Batch processing Sputum, AFB, Viral, Fungal Cary-Blair Stool
Level 1 specimens are collected from individuals with potentially life-threatening illnesses (2nd choice: Alkaline Peptone Water)
Level 2 specimens may quickly deteriorate or may have overgrowth of contaminating organisms JEMBEC:
Genital specimens N. gonorrhoeae
CAP + CO2-generating tablet
ALL SPECIMENS ARE PLACED IN A STERILE SCREW-CAP CONTAINERS EXCEPT FOR THE FOLLOWING: Transgrow Neisseria
Todd-Hewitt and LIM Broth
COLLECTION MEDIUM SPECIMEN Vaginal swab S. agalactiae
(Modified Todd-Hewitt)
Pre- Stuart’s or Armie’s Upper respiratory tract: nose, nasopharynx, throat, larynx Hanks Balanced Salt Solution
moistened JEMBEC Genitourinary tract: vaginal canal, cervix (non-pregnant), with Bovine Albumin, Stuart, Viral transport media
swab LIM Broth: vaginal swab in pregnant urethra, prostate secretions Leibovitz-Emory
placed in Cary-blair Rectum: rectal swab
Transport Outer ear It is a SEMI-SOLID media and contains CHARCOAL is added to transport media to:
medium Superficial lesion: abscess, wound, pustule, ulcer  absorb the fatty acid present in cotton swab which is toxic to some bacteria (e.g., N. gonorrhoeae, B. pertussis )
Clean Leak-proof container  neutralize bacterial toxins and other inhibitory substances
Stool; use transport medium if transport exceeds 1 hour
(plastic or wxed cardboard)  maintains pH
Bedside Plating Corneal scrapins
Clean screw-top tube Hair, nails, skin scrapings SWAB

IDEALLY, SPECIMENS SHOULD BE TRANSPORTED TO THE LABORATORY 2 HOURS OF COLLECTION EXCEPT: Dacryon and Rayon is recommended for bacteria and viruses

IMMEDIATELY < 15 MINUTES WITHIN 1 HOUR WITHIN 24 HOURS COTTON: toxic to Neisseria, but good for virus
Prostatic fluid in glass Sterile body fluids (CSF, Unpreserved stool Preserved stool CALCIUM ALGINATE: toxic to virus, good for Neisseria
tubes (within 2 hours if amniotic, peritoneal, bile, SPECIMEN TYPE SWAB TYPE DO NOT USE
prostate fluid swab) synovial, pericardial, Unpreserved urine (within Rectal swab Throat culture CALCIUM ALGINATE, DACRON
pleural, aqeous/vitreous 30 minutes) Genital specimen DACRON, RAYON Wooden cotton swab
Suprapubic aspirate fluid from eyes) except Preserved urine Urogenital COTTON SWAB with Charcoal
blood Virus DACRON Calcium alginate, Wooden swab
Bone Gastric biopsy Hair, nails, skin scrapings Best Overall Swab FLOCKED NYLON
Gastric aspirate (infant) C. trachomatis and HSV are INHIBITED by wooden shat and SENSITIVE to cotton swab

Tissue biopsy SWAB IS NOT RECOMMENDED FOR FUNGI AND ANAEROBES

Corneal scrapings
PRESERVATIVE

Foreign bodies  Anticoagulants or preservatives are added to maintain accurate colony count; should any delay occur, 0.025-
(intrauterine device, IV 030%% Sodium Polyanethol Sulofnate (SPS) may be added (especially for blood culture)
catheter, surgical pin) ► It prevents clotting
► It inhibits phagocytosis and complement activation
► It neutralizes the activity of aminoglycoside antibiotics
► It neutralizes the bactericidal effect of plasma

Page 21 of 40
 SPS DISADVANTAGE: inhibits Neisseria, Gardenrella vaginalis, Peptostreptococcus anaerobius,
Streptobacillus monoliformis CRITICAL VALUES
(The inhibitory effect can be counteracted by 1% Gelatin)
 Heparin, EDTA, and Citrate should be avoided because they may suppress the growth of organisms
► The information on the label does not match the information on the requisition or the specimen is not Positive blood
o Heparin may inhibit gram-positive bacteria and yeast, however is used in VIRAL and MYCOBACTERIUM
culture
CULTURE
► Positive cerebrospinal fluid gram stain or culture
o Citrate diminishes gram-positive cocci activity
► Positive cryptococcal antigen test or culture
 WHITE TOP TUBE with EDTA: used only when PCR is the procedure of choice ► Positive blood smear for malaria (STAT)
 BORIC ACID for Urine ► Positive acid-fast smears or positive Mycobacterium culture
 POLYVINYL ALCOHOL (PVA) and BUFFERED FORMALIN for stool ova and parasite (O&P) examination ► Streptococcus pyogenes from a sterile site
► Streptococcus agalactiae or herpes simplex virus from genital site of a pregnant woman at term
 Specimens requiring anticoagulant: blood, bone marrow aspirate, synovial fluid ► Detection of significant pathogen (i.e., Bordetella pertussis, Brucella, Legionella, Anthrax)

!! REPORT THE RESULT IMMEDIATELY !!

TEMPERATURE

Specimens suspected to contain ANAEROBE SHOULD NOT BE REFRIGERATED

STORAGE
Anaerobic culture, sterile body fluids, genital specimens,
RT at 22-25oC
swabs, hair, nail, skin scraping
37oC in < 24 hours
CSF for routine studies
NEW: 22oC (RT)
Viral specimens 4oC for up to 3 days
Urine, stool, viral specimens, sputa, swabs, foreign devices (catheters),
4°C
gastric biopsy/lavage, BAL
Serum for serologic studies frozen for up to 1 week at –20° C
Tissues or specimens for long-term storage frozen at –70° C

REJECTION OF UNACCEPTABLE SPECIMENS

► The information on the label does not match the information on the requisition or the specimen is not
labeled at all (patient’s name or source of specimen is different)
► The specimen has been transported at the improper temperature
► The specimen has not been transported in the proper medium (e.g., specimens for anaerobic bacteria
submitted in aerobic transports).
► The quantity of specimen is insufficient for testing (the specimen is considered quantity not sufficient [QNS])
► The specimen is leaking.
► The specimen transport time exceeds 2 hours post-collection or the specimen is not preserved
► The specimen was received in a fixative (formalin), which, in essence, kills any microorganism present
► The specimen has been received for anaerobic culture from a site known to have anaerobes as part of the
normal flora (vagina, mouth)
► The specimen is dried
► Processing the specimen would produce information of q questionable medical value (e.g., Foley catheter tip).

It is important to always talk to the requesting physician or another member of the health care team before
discarding unacceptable specimens

Mislabeling of a specimen or requisition may be corrected by having the person who collected the specimen
with corrective measures following laboratory protocol

Some mislabeled specimens may undergo special testing (sub-optimal) for considerations especially those
obtained from invasive procedures

Page 22 of 40
 CLINCAL SPECIMENS CSF

BLOOD  CONTAINER: Sterile, screw-cap tube or anaerobic transporter

 Patient Preparation: skin is disinfected before aspirating specimen, in fetus position
 Collect before the height of fever (at 37.5 oC or 37.8oC bacteria are dead)  Consider rapid testing (e.g, gram stain, cryptococcal antigen) and transported in < 15 minutes
o Highest concentration of microbe: shortly before and during height of fever o Specimen is subjected to cytocentrifugation; sediment is used for culture and smear preparation
o Use Acridine Orange if there is no cytocentrifuge
 DISINFECTION: (1) 70% alcohol then with Betadine or (2) Clorhexidine
 APPLICATION OF ANTISEPTIC: (1) Circular – Bailey and Scott’s (2) Up and down – CLSI  Usually collected through Lumbar Tap between L3 and L4 in 3-4 tubes, at least 1 mL and not >10 mL
 INCUBATION: 35-37oC o #2: Microbiology at RT
 DRAW 2-3 Sets (Max of 3) at least 1 hour apart; do not draw more than three (3) sets in a 24-hour period o 3#4: used for better exclusion of skin contaminants
o In cases of remote areas with unavailable blood culture bottles, use of SPS (Yellow top) is used o 1 tube: Microbiology > Hematology > Chemistry > Other tests
► 2-3 specimens from different sites drawn at least 1 hour apart
► Adult: minimum of 10 mL (1:10 dilution of blood to culture media) is distributed to the following:  STAINS
» AEROBES: 5 mL TSB or BHI o Routine: Gram stain, India ink
» ANAEROBES: 5 mL Thioglycollate broth (should be colorless, boil to drive off oxygen if o Parasitic meningitis: Giemsa, H&E stain
appears too pink)
 Often the priority (processed immediately) and collected to rule out Meningitis
o In cases of delayed processing, hold at 37oC and transported at RT
 CONTAINER: Blood Culture set (Aerobic and Anaerobic) collected on opposite arms; Blood: Broth ratio = 1:10
Vacutainer tube with SPS STORAGE: incubate at 37oC in < 24 hours for routine testing except for viruses (held at 4oC for up to 3 days)
ADULT ≥ 20 mL; 10 mL for Aerobic and Anaerobic
PEDIATRIC 1-20 mL (weight-dependent)
CHILDREN 1-5 mL MENINGITIS
INFANTS 0.5-1.0 mL
 Neonates (birth to 1 month): Streptococcus agalactiae
EMERGENCY 40 mL at one collection; immediate antibiotic therapy administration
 Children (1-5 years old): Haemophilus influenzae Serotype B – CAP, Bacitracin-CAP
 5-29 years old: Neisseria meningitidis - TMA
 ANTIMICROBIAL REMOVAL DEVICE (ARD) and THIOL BROTH  Adult (>29 years old): Streptococcus pneumonia – BAP, Gentamicin-BAP
o Used in patients on antimicrobials to neutralize medication and remove them before culture  Elderly or Immunocompromised (including infants): Listeria monocytogenes
PENCILLINASE Penicillin
RECALL: Cryptococcus neoformans resembles lymphocyte in CSF
MAGNESIUM SULFATE Tetracycline
Coccidioides imitis increased eosinophil in CSF
PARA-AMINO BENZOIC ACID (PABA) Sulfonamide
Blastomyces myelin globules in sputum
 ISOLATOR TUBES (CLOT –LYSIS SYSTEM)
o For centrifugation and concentration of microbes (bacteria, fungi) from blood and bone marrow  Addition of THIOGLYCOLLATE after CSF collection from a shunt is done to allow growth of facultative
particular for Brucella or other intracellular organisms anaerobes, strict aerobes, and anaerobes
 Routine Culture: BAP, CAP, Thayer-Martin Agar
ROUTINE BLOOD CULTURE  CSF collected from shunt: BAP, CAP, THIOGLYCOLLATE
MOST
LEPTOSPIRA BRUCELLA  For the isolation of:
BACTERIA
6-8 weeks 3-4 weeks  Neisseria meningitidis
5-7 days  Streptococcus pneumoniae, Streptococcus agalactiae
(1 week) Blood is collected in early infection (urine in the 2 nd week) Normal flora in animals;  Listeria monocytogenes
Serology is the GOLD STANDARD for Leptospirosis detection causing febrile disease in man  Haemophilus
SIGNS OF GROWTH: turbidity, bubbles, clots, hemolysis  Escherichia coli
 Staphylococcus aureus
 POSITIVE (+) CULTURE:
1. Gram stain and subculture
2. If one organism is observed, proceed to AST
3. If polymicrobial, culture in enriched and selective media for suspected pathogen
4. If negative (-) for grams stain, stain with Acridine orange (all sterile body fluids for nucleic acid)

BACTEREMIA SEPSIS
Presence of bacteria in the blood Bacteria increasing in numbers in the blood
causing harm to the patient
Most common cause: Gram (+) cocci

Page 23 of 40
 RESPIRATORY TRACT SPECIMEN
BARTLETT’S CLASSFICATION (SPUTUM vs. SALIVA)
 Most abundant normal flora in the upper respiratory tract: Alpha-hemolytic Strep
 Most common pathogen: Beta-hemolytic Strep (S. pyogenes) Assesses the quality and for scoring of sputum, the # neutrophils and epithelial
cells/LPF is enumerated.
 Collected for streptococcal infection containing either:
 S. pyogenes: major throat pathogen ≤0 lack of inflammation or the presence of saliva
 Viridans strep >1 inflammation or infection
 C. diptheriae
 B. pertussis:
 N. meningitidis : carrier state # of neutrophils per low-power field Grade
# of epithelial cells
Grade
THROAT SWAB per low-power field
 N. meningitidis a normal flora (nasopharynx or oropharynx - NPS) or pathogen Fewer than 10 0 <10 0
(meningitis - CSF) 10-25 +1 10-25 -1
o Sensitive to COLD TEMPERATURE and toxic to COTTON SWAB Greater than 25 +2 Greater than 25
-2
containing fatty acids; use Dacron or Calcium alginate Mucus +1
o ENDOTRACHEAL ASPIRATE (ETA): specimen to collect when patient is on ACCEPTABLE: <10 SECs/LPF and >25 PMNs/LPF
a ventilator or intubated ORAL SECRETION (SALIVA): >10 SECs/LPF and <25 PMNs/LPF
RESPIRATORY SECRETION (SPUTUM): presence of Alveolar macrophage (>25/LPF)
Calcium Alginate is toxic to viruses as it will inhibit the replication; Dacron can be used
SPUTUM COLOR

 CONTAINER: Sterile, screw-cap container BLUE-GREEN P. aeruginosa

 Processed to diagnose Lower Respiratory Tract Infections (LRTIs) (e.g., RUST-COLORED S. pneumoniae
pneumonia, bronchitis, and tuberculosis) CURRANT-JELLY-LIKE K. pneumoniae
 Sputum is a non-sterile specimen that is why it is needed to evaluate its quality (to HEMOPTYSIS (BLOODY) M. tuberculosis, Nocardia, Histoplasma, Paragonimus
check if it is sputum or saliva) OVA IN SPUTUM Paragonimus
LARVA IN SPUTUM Migrating (ASH) – Ascaris, Strongyloides, Hookworms
 Patient must be instructed to brush their teeth, gargle with water prior to collection
and cough deeply into container
 Induced sputa on pediatric or uncooperative patients can be done with:
► Aerosolized droplets of 15% NaCl + 10% Glycerin for 10 minutes or until a
strong cough is reflexed)

SPECIMEN COLLECTION
Bailey and Scott’s DOH
1 sputum specimen collected in 3 days 2 specimens collected in 1 day

1st collection: first morning sputum

2nd collection: random  Moistened swab inserted 1-2 cm in the nares/nose detecting the carrier state of S.
aureus and MRSA
SPUTUM (+): 2 out of 3 (+): 1 out of 2 NASAL SWAB  Transported to the laboratory < 2 hours at RT
 Storage before processing at 24 hours at RT
 Hepatitis B virus (HBV) has not been islated in sputum
 Add special media for C. diphtheria, B. pertussis, Chlamydia, Mycoplasma
 The specimen of choice for M. tuberculosis (slow grower) identification, sputum must  Flexible swab inserted through the nose and into posterior nasopharynx and then
undergo decontamination and digestion PHARYNGEAL SWAB rotated for 5 seconds
o MTB is a BSL-3 pathogen ∴ culture must be done using BSC Class II  Detects the carrier state of N. meningitidis and detection of B. pertussis
o AFB stains must be read at least 300 OIF before reporting negative (-);
taking up 3-5 minutes per slide Provides the best specimen for Upper Respiratory Tract

Throat or epiglottis swab on enriched CAP with S. aureus across

 A direct gram stain is performed to determine the quality of specimen; thumb-sized H. influenzae
coiled smear (2 cm x 3 cm) NASOPHARYNGEAL the inoculum
o Acceptable specimens are cultured on SBA, MAC, CAP SWAB N. meningitidis Enriched CAP
B. pertussis Charcoal Cephalexin Medium (Regan Lowe)
 SPUTUM PATHOGENS:
C. diphtheria CTBA
 S. pneumoniae
 K. pnuemoniae MRSA CHROM agar
 M. pneumoniae  Procedure can best be done on ambulatory patients who are able to follow
 S. aureus AEROSOL (SALINE) instructions
 P. aeruginosa INDUCTION  Aerosol-induced sputum specimens have been collected from children as young as 5
 H. influenza
years of age
 L. pneumophila
Page 24 of 40
  Used to collect sputum from patients who may have swallowed sputum at night GASTROINTESTINAL SPECIMEN
 Procedure is limited to senile, non-ambulatory patients
GASTRIC LAVAGE
 Specimen of choice for children younger than 3 years of age  CONTAINER: Clean, leak-proof container either plastic or wax cardboard (1-2g of stool)
 Patients who fail to produce sputum by aerosol induction  STORAGE after culture: refrigerator
 Collected and submitted by medical personnel
BRONCHIA LAVAGE,  SPECIMENS OF CHOICE FOR DETECTING NONTUBERCULOUS SPECIMEN COLLECTION SPECIMEN TRANSPORT MEDIA
WASHINGS, MYCOBACTERIA AND OTHER OPPORTUNISTIC PATHOGENS IN PATIENTS Cary-Blair
3 specimens EMB
BRUSHINGS WITH IMMUNE DYSFUNCTION Bacteria MEDIA Glycerol- Differential
(1 each day) MAC
 P. carinii (P. jiroveci) causes respiratory infections among immunosupressed saline
 Endotracheal aspirates (ETAs) from MECHANICALLY VENTILATED adult patients Parasite
3 specimens in Preserved within 24
Selective
HEA
can be screened by Gram stain 10 days stool hours at RT SSA
ENDOTRACHEAL
 Criteria used to reject ETAs from adult patients include: Amoebiasis
6 specimens in Unpreserved
<1 hour at RT Enrichment Tetrathionate
ASPIRATES (ETA) 10 days stool
 >10 squamous epithelial cells per low-power field
 no organisms seen under oil immersion (1000×)
WAITING TIME PRIOR COLLECTION
URINE Bismuth or Barium intake 3-7 days
Antibiotic intake 2 weeks
 CONTAINER: Sterile, screw-cap container
 The specimen to rule out repeated UTI PRESUMPTIVE CONFIRMATORY
Biochemical (e.g., Oxidase) Serotyping
Specimen of choice for bacterial culture
MIDSTREAM CLEAN CATCH
(Clean-voided Midstream – CVS) Females/Males clean genitals with soap and water, hold labia
CAUSES OF DIARRHEA
apart/retract foreskin, void and collect mid-portion
► ROTAVIRUS: most common cause of diarrhea and gastroenteritis in children
Anaerobic culture; needle aspiration above the symphysis
SUPRAPUBIC URINE ► NOROVIRUS (NORWALK): most common cause of diarrhea in adutlts
pubis through the abdominal wall into the full bladder STOOL
FIRST MORNING URINE Molecular studies ► C. difficile: common cause of diarrhea among hospitalized patients (CLINDAMYCIN is
Catheter inserted into bladder, allow first 15 mL to pass before the first antibiotic to note this condition)
STRAIGHT CATHETER (IN and OUT)
collecting the remainder
INDWELLING CATHETER (FOLEY) Aspirate 5-10 mL of urine with needle and syringe
CAUSES OF GASTROENTERITIS
► Shigella
CAUSES OF UTI ► Salmonella
► # 1: E. coli - UPEC (90% of the cases) ► Campylobacter jejuni
► Young female: S. saprophyticus
► E. coli O157:H7
► Hospitalized patients: Enterococci
► Other causes: Klebsiella, Enterococcus faecalis ► Yersinia enterocolitica
► Clostridium difficile- tested for cytotoxin
► Vibrio
 COLONY COUNT: calibrated loop (1-10 uL) is used for inoculation to determine the dilution factor; [erform
colony count using calibrated loop:
 FOLLOW UP: Shiga toxin assay as recommended by CDC
# of colonies x dilution factor = colony count/mL of urine
1 uL loop  Do NOT perform routine stool cultures for patients whose length of stay in the hospital exceeds 3
1000 DF
10 uL loop 100 DF days and whose admitting diagnosis was not diarrhea; should be tested for Clostridium difficile
UTI colony count is >100,000 CFU/mL or 105 CFU/mL
 Routinely screen for:
 SARS-COV2 has not beed isolated in urine and Legionella pneumophila antigen however can be detected  Salmonella
 Shigella
 Must be cultured within one hour (refrigerated if not processed but not longer than 24 hours) and preservation in  Campylobacter
BORIC ACID will maintain accurate colony count
 Specification for Vibrio, Aeromonas, Plesiomonas, Yersinia, E. coli O157:H7

 Usually held in Cary-Blair, best specimen for viral culture

RECTAL  COLLECTION: feces should be visible on the swab and is inserted
SWAB ► 1-1.5 cm past anal sphincter
► 2-4 cm from anal verge, rorate 3x

Page 25 of 40
 ABSCESS (WOUND, LESION, PUSTULE, ULCER)
GASTRIC  Place in transport medium and transported within 1 hour CONTAINER PATIENT PREPARATION COLLECTION
BIOPSY  For the rapid urease test and culture of H. pylori Swab along the leading
edge of the wound
SUPERFICIAL (SKIN) Aerobic swab moistened with
 Best specimen for infants; collected early in the morning before rising and first meal ABSCESS Stuart’s or Amie’s medium Wipe area with Add CAN if smear
GASTRIC o For the examination of AFB for TB diagnosis among infants sterile saline or 70% alcohol suggests mixed gram
ASPIRATE  Transported within 15 minutes (+) and gram (-) flora
 Sample must be neutralized with Sodium bicarbonate (NaHCO3) within 1 hour INTERNAL (DEEP)
Anaerobic transporter
Aspirate material from
ABSCESS wall or excise tissue

GENITAL TRACT SPECIMENS

SPECIMEN FOR MOLECULAR TECHNIQUES
 TRANSPORT MEDIA: JEMBEC or Gono-Pak
 Collected for the diagnosis of cervicitis and Venereal diseases or STDs  Does not always require the detection of viable organisms
 The timing and collection devices used remain critical to the successful detection of nucleic acids
 Mostly fastidious organisms requiring the use of CAP
USE DO NOT USE
 Vagina contains normal flora that changes with age: WHITE TOP TUBE (EDTA) for plasma Heparinized plasma inhibits polymerase enzyme
o Lactobacillus: during childbearing age Plastic swab is recommended for collection from Calcium alginate swab with aluminum shaft
o Staphylococci and Corynebacteria: earlier and late in life mucosal membranes interfere with nucleic acid amplification

 SPECIMENS: FLOCK NYLON SWAB

► Cervical swab: female
► Urethral swab: male  SPECIMEN: first voided urine transported in ice or broth provided
► Rectal swab
► Throat swab

URETHRAL SWAB is the best specimen for both male and female for STI detection SPECIMEN PREPARATION

 Molecular techniques: commonly employed for N. gonorrhoeae and C. trachomatis detection ► Homogenization (grinding) of tissue
o FIRST MORNING URINE: the specimen of choice for NAAT ► Concentration by centrifugation or filtration of large volumes of sterile fluids (e.g., ascites or pleural)
► Decontamination for Legionella and Mycobacteria
 Neisseria gonorrhoeae and Bordetella pertussis (not an STD) are SENSITIVE to: ► Vortexed (Mixed) for swab specimen in 0.5-1 mL saline or broth for 10-20 seconds to dislodge material from
► Cotton swab; use Dacron or Rayon instead fibers
► Cold temperature; do bedside plating and incubate immediately
► Drying; use moist swab To visualize bacterial cells by light microscopy, a minimum concentration of 10 5 cells/1mL of specimen is required
► Fatty acids; add charcoal to the transport medium

 To detect the presence of:

 Neisseria gonorrhoeae: second most common cause of STI
 Gardnerella vaginalis
 Trichomonas vaginalis
 Chlamydia trachomatis: most common cause of STI (best specimen: Ure
 Treponema palladium
 Herpes Simplex Virus

CORNEAL SCRAPING

 Bedside plating on BHI, SDA (fungi), BAP, CAP, Thioglycollate streaked in a “C” pattern
 Smear on 10mm frosted ring slides

Page 26 of 40
 BACTERIAL IDENTIFICATION STAINING TECHNIQUES
Simple (aka Direct) uses one dye; all organisms have the same color
METHODS OF STUDYING MICROORGANISMS Differential uses two different dyes; commonly employed in gram and acid fast staining
Living State (Unstained) Fixed State (Stained) the background is stained leaving the capsule unstained
Indirect (aka Negative, Relief)
Wet Mount Preparation: specimen is mixed with NSS STEPS (India Ink test or Borris method and Nigrosin Method)
1. Bacterial Smear Preparation Special used to demonstrate special features of the cell
Hanging Drop Preparation: uses concave slide to 2. Fixation: using heat or 70-95% alcohol (methanol)
demonstrate the motility of an organism 3. Staining GRAM STAINING

MORPHOLOGY  Developed by Danish bacteriologist Hans Chrsiitan Gram in 1884

 The following should be considered: size, shape, arrangement, motility, and staining characteristics  Stains the tecichoic acid present in cell walls of a gram-positive (+) bacteria
 Not applicable to:
STAINING is the process of artificially coloring the organism with dye with the following purposes:  organism that exists almost exclusively within host cells (e.g. Chlamydia)
► To observe and appreciate the appearance of bacteria  those lack cell walls (e.g. Mycoplasma and Ureaplasma)
► To differentiate one organism from the other  those of insufficient dimension to be resolved by light microscopy (e.g. Spirochetes)
► To reveal the chemical nature of bacteria

STAINS GRAM STAINING GENERAL RULE

 HISS
CAPSULE  India Ink (Nigrosin)  All cocci are gram (+) except Neisseria, Veilonella, and Moraxella
 Other: Muir, Anthony’s, Tyler, Welch’s, Grins
 Gram’s  All bacilli are gram (-) except Mycobacteria, Corynebacteria, Clostridia, Bacillus, Erysipelothrix,
 Acid fast: Ziehl-Neelsen, Kinyoun, Pappenheim, Baumgarten, Fite Faraco Lactobacillus, Listeria, Actinomyces, Nocardia, Gardnerella, Arcanobacterium
CELL WALL  Modified Acid fast
 Auramine-Rhodamine  All true gram (-) organisms have ENDOTOXIN (Lipid A) as part of their outer membrane
» bright yellow or orange against green background for Mycobacterial cells
 Schaeffer-fulton  Higher forms or organisms considered gram (+)
» Primary stain: Malachite Green ► Yeasts
» Counterstain: Safranin ► Actinomyces
SPORES » ENDOSPORES appear green within pink- or red- appearing bacterial cells  NOT GRAM STAINED:
 Dorner’s ► Chlamydia, Rickettsia: INTRACELLULAR
 Wirtz and Conklin ► Mycoplasma, Ureaplasma: NO CELL WALL (gram negative stained but without endotoxin)
 Other: Heat and Acetic acid Method ► Spirochete: CAN’T BE RESOLVED BY BRIGHT FIELD
 Leifson ► Legionella, Spirochetes: SILVER IMPREGNATION TECHNIQUE is most useful
 Gray’s
FLAGELLA
 Fisher and Conn
 Other: Loeffler’s, Caesars Gil, Van ermenger PURPOSE REAGENT GRAM (+) GRAM (-)
 Acridine Orange: confirmation of gram (-) stains, particularly for Mycoplasma Primary Stain CRYSTAL VIOLET Purple Purple
NUCLEIC ACID » (+) bright orange Mordant
 Feulgen (enhances the affinity of the
POLAR BODIES  Wayson 1o stain to the cell wall and GRAM’S IODINE Purple Purple
 Levaditi create an insoluble complex of
SPIROCHETES  Fontana-Tribondeau the dye and cell wall)
 Warthin starry Decolorizer
95% ETHANOL or ACETONE ALCOHOL Purple Colorless
 Gimenez (the most crucial step)
RICKETTSIA  Macchiavelo Secondary Stain SAFRANIN Purple Red
 Giemsa Bacterial smear is subjected to (1) heat fixation or (2) slide warming
 Burke’s Modification of Gram Stain
 Leoffler’s Alkaline Methylene Blue (LAMB)Neisser LPO → OIO: Once stained, the smear is examined using the 10x objective (100x magnification). Scan the slide for
METACHROMATIC
 Albert’s white blood cells, epithelial cells, debris, and larger organisms (fungi, parasites) followed by examining
GRANULES
 Ljubinsky through oil immersion (1000x magnification) lens.
 Other: Lindergan, Ponder
 Calcofluor white: bright green apple or blue-white for CHITIN in fungal cell wall  Bordetella pertussis is left in Safranin for (1) 2 minutes or (2) replace safranin with 0.2% Basic Fuchsin
CHITIN
 Lactophenol cotton blue: stain used in Aman’s medium  Legionella have poor-staining characteristics, 0.1% BASIC FUSCHIN is used as secondary stain

Page 27 of 40
 COMPARISON BETWEEN GRAM-POSITIVE (+) AND GRAM-NEGATIVE (-) BACTERIA
DECOLORIZATION Properties GRAM (+) GRAM (-)
Outer membrane composed of lipid,
► GRAM POSITIVE (+): Crystal violet – Iodine – Teichoic acid MgRNA-complex = PURPLE THICK cell wall composed of
CELL WALL protein, and lipopolysaccharide with
► GRAM NEGATIVE (-): Lipids are highly soluble to alcohol peptidoglycan
an inner thin peptidoglycan
Spherical, rods, ovals, straight,
POSITIVE CONTROL: Staphylococcus aureus SHAPE Spherical, rods, or filaments
curve, helical
NEGATIVE CONTROL: Escherichia coli TEICHOIC ACID PRESENT ABSENT
ENDOSPORES Present in some groups ABSENT
PERIPLASMIC SPACE ABSENT PRESENT
COCCI FLAGELLAR STRUCTURE Two (2) rings in basal body Four (4) rings in basal body
AEROBIC ANAEROBIC INHIBITION BY BASIC DYES HIGH LOW
GRAM (+) GRAM (-) GRAM (+) GRAM (-)
Staphylococci Peptococcus
Neisseria
Micrococci Peptostreptococcus Veilonella RECALLS
Moraxella
Streptococci Sarcina
► GRAM STAIN: most important diagnostic tool in treating patients with infections in the emergency department
BACILLI ► TOLUIDINE BLUE O: rapid tool and best for fungi identification in times of emergency
AEROBIC ANAEROBIC
GRAM (+) GRAM (-) GRAM (+) GRAM (-)
Acinetobacter
Aeromonas
Bacillus Alcaligenes
Corynebacterium Bordetella
Actinomyces
Erysipelothrix Brucella Fusobacterium
Clostridium
Listeria Enterobacteriaceae Bacteroides
Propionibacterium
Mycobacterium Francisella
Nocardia Legionella
Pasteurella
Pseudomonas

GRAM STAINING MODIFICATION

Hucker’s Modification Burke’s Modification
STEPS Metachromatic Granules
(FUNGI) (METACHROMATIC GRANULE)
Methyl violet – Crystal Violet –
Primary Stain Crystal Violet
Sodium bicarbonate Ammonium oxalate
Mordant Gram’s Iodine
Sodium bicarbonate –
Decolorizer Acetone Acetone-Alcohol
Ether-acetone
Secondary Stain Safranin O or Safranin Y Safranin Safranin

ERRORS IN GRAM STAINING

Gram (+) becomes Gram (-) Gram (-) becomes Gram (+)
 Over-decolorization  Under-decolorization
 Old dye  Thick smear
 Use of acidic iodine as mordant
 Penicillin (loss of cell wall integrity)
 Omit mordant

Page 28 of 40
 ACID FAST STAINING KINYOUN’S (COLD METHOD)
STEPS REAGENT TIME ACID FAST NON-ACID FAST
 Stains the MYCOLIC ACID (aka Hydroxymethoxy acid) in the cell wall of bacteria Primary Stain CARBOL FUCHSIN Red Red
 ACID FAST ORGANISMS: Chemical:
5 minutes
 Mycobacteria Mordant TERGITOL or 10% Red Red
 Nocardia (partially acid fast) PHENOL
 Some parasites (Cryptosporidium, Isospora, Cyclospora) ACID ALCOHOL
Decolorization 2 minutes Red Colorless
(3% H2SO4 + 95% Ethanol)
 Mycobacterium appears as “GRAM GHOST” or “GRAM NEUTRAL” after gram staining, failing to take up the METHYLENE BLUE or
stains due to the mycolic acid Secondary Stain 1-3 minutes Red Blue or Green
MALACHITE GREEN
o HARD TO STAIN because it resists decolorization within an acid environment hence “ACID-FAST” 10% Phenol is already included in the Carbol fuchsin

 AFB SMEAR SIZE (DOH): “Thumb-sized” 2 cm x 3 cm MODIFIED KINYOUN METHOD for Partially Acid Fast Organisms
STEPS REAGENT TIME ACID FAST NON-ACID FAST
CLINICALLY RELEVANT AEROBIC ACTINOMYCETES Primary Stain CARBOL FUCHSIN Red Red
CELL WALL with MYCOLIC ACID CELL WALL without MYCOLIC ACID Mordant Chemical: TERGITOL Red Red
Mycobacterium ACID ALCOHOL
Nocardia Decolorization Red Colorless
Streptomyces (1% H2SO4 + 70% Ethanol)
Rhodococcus Secondary Stain METHYLENE BLUE Red Blue
Actinomadura
Gordonia
Dermatophilus Particularly used for partially acid fast parasites that resembles spores
Tsukamurella
Nocardiopsis
Corynebacterium
Oerskovia AURAMINE-RHODAMINE (TRUANT’S) STAINING METHOD – FLUORESCENT ACID FAST
STEPS REAGENT TIME ACID FAST NON-ACID FAST
More resistant to toxic substances including acids
Primary Stain AURAMINE-RHODAMINE
Mordant NONE
Decolorization 0.5% ACID ALCOHOL
WAYS TO FACILITATE ACID FAST ORGANISMS
POTASSIUM
Secondary Stain Yellow Fluorescence NO fluorescence
PERMANGANATE (KMNO4)
 Steaming process
Aurum meaning “golden yellow”
 Addition of wetting agent (TERGITOL) prior to the stain solution
OTHER MODIFICATIONS
 Increasing concentration of the accentuator (PHENOL) and basic fuchsin
METHOD NOTES POSITIVE (+) NEGATIVE (-)
 Prolonging the contact of stain with the material
Differentiates Mycobacterium
Pappenheim’s smegmatis from Mycobacterium M. tuberculosis M. smegmatis
tuberculosis
ZIEHL-NEELSEN KINYOUN AURAMINE-RHODAMINE
Differentiates Mycobacterium
PREFFERED and M. leprae M. tuberculosis
Routine AFB staining BEST TISSUE AFB STAIING Baumgarten’s leprae from Mycobacterium
MOST SENSITIVE AFB STAINING METHOD (RED) (BLUE)
method of choice; METHOD OF CHOICE tuberculosis
(no mordant due to the strong affinity of
BEST ROUTINE METHOD (no application of heat)
Auramin O and Rhodamine B on the mycolic acids)
FITE-FARACO: uses Hematoylin as counter stain
SPENGLER’S METHOD: intended for colorblind individuals; AFB appears black
HPO → OIO: Smear is screened at 400 magnification and confirm all suspicious (e.g., red) organisms at 1000
magnification (OIO)

ZIEHL-NEELSEN STAIN (HOT METHOD)

STEPS REAGENT TIME ACID FAST NON-ACID FAST
Primary Stain CARBOL FUCHSIN Red Red
Physical: 4-5 minutes
Mordant Red Red
HEAT or STEAM
ACID ALCOHOL
Decolorization 2 minutes Red Colorless
(3% HCl + 95% Ethanol)
METHYLENE BLUE or
Secondary Stain 1 minute Red Blue or Green
MALACHITE GREEN
Slide is flooded with the primary stain and heated until boiling. Then it is left to cool prior flooding with Acid alcohol

Page 29 of 40
 STUDY OF CULTURAL CHARACTERISTICS TYPES OF CULTURE MEDIA

Culture is/are microorganissms in specimen can be cultivated and grown in artificial media (a culture medium containing A. According to Physical State
nutritional requirements needed for bacterial growth)
STATE SIGN OF GROWTH SOLIDIFYIG AGENT EXAMPLES
TYPES OF CULTURE MEDIUM  Nutrient Broth
PURE CULTURE MIXED CULTURE STOCK CULTURE none;  Brain Heart Infusion
Liquid Turbidity
for rapid culture  Alkaline Peptone Water
for identification and AST of one organism
 Thioglycolate
Ways of isolation: 0.5-1% agar;  SIM
Semi-solid
► Streak/Spread plate method: BEST more than one species for ATCC for motility and transport media  Gelatin Media
► Pour plate method LIQUEFIABLE (2-3%)
2-3% agar
► Selective medium  SSA
► Animal inoculation  BAP
Colonies and
Solid  MAC
Color Change Firm Agar (6%): often used to
MEDIA PREPARATION NON-LIQUEFIABLE (non-agar)
prevent the swarming of Proteus
 Chopped Meat (Anaerobes)
Water-Agar-Water and Clostridium
 Rice Medium (for fungi)
Use of deionized water is recommended but distilled water is acceptable
Biphasic Solid (bottom) and Liquid (top) Castañeda for Brucella
Plated (Petri Dish) Tube (Test Tube)
1. Weigh agar using analytical balance 1. Weigh agar using analytical balance
B. According to Composition
2. Dissolve in deionized or distilled water 2. Dissolve in deionized or distilled water
3. Sterilize in autoclave 3. Dispense
SYNTHETIC NON-SYNTHETIC
4. Dispense 4. Sterilize in autoclave TISSUE CULTURE
(DEFINED) (COMPLEX)
STERILIZATION can be done through autoclave (121oC for 15 minutes) or membrane filtration (0.2-0.45 um)
 All components  Composed of  Uses living cell or virus wherein
are known unknown substance CYTOPATHIC EFFECT is observed
Cooling temperature for distribution of media to plate is 55-60 oC with 25 mL per plate.  Very useful for
isolation of bacteria Chick Embryo: Rickettsia, Viruses
3 mL LIQUID BROTH HeLa cells: cervical cancer
McCoy cells: mouse cell line for Chlaymidia
6 mL AGAR SLANT
15-20 mL AGAR PLATE A549: lung carcinoma
Vero cell: African Green Monkey Kidney cells
Hep2 cells: laryngeal cancer cell (ANA)
Agar is usually derived from red algae (Genus Gelidium) which:
► SOLIDIFIES at <50oC (Old: 40-50oC)
► MELTS at ≥ 95 oC (Old: 80-90oC) C. According to Purpose

SIMPLE Support the growth of most NON-FASTIDIOUS BACTERIA

(GENERAL, whilst giving no growth advantage to any group of bacteria
BASAL,
SUPPORTIVE) Ex. Nutrient agar, Nutrient broth Trypticase Soy agar or broth,
ISOLATION Sabouraud’s dextrose agar (for fungi)

Used for growing FASTIDIOUS ORGANISMS; for general isolation with added nutrient
(e.g. blood from sheep > horse > rabbit, serum, peptone, and vitamins)
ENRICHED
Ex. BAP (5% defibrinated sheep blood), CAP (heated blood) BCYE (yeast extract)
(NON-
Human BAP (X and V factors)
SELECTIVE
ISOLATION)
Note: human blood is not preferred because it contains non-specific inhibitors
MEDIA
(citrate – inhibits B-hemolytic Strep and dextrose – alters the hemolytic pattern)

5% defibrinated sheep blood > horse blood (Haemophilus) > rabbit

Page 30 of 40
 BACKUP BROTH: 0.075% agar and thioglycollate (reducing agent) creating anaerobiosis (boiling drives off oxygen)
Used for specimens that generally have a mixture of organism (contaminants);
inhibits the growth of one organism while enhancing that of another organism GROWTH PATTERN IN BROTH
(by providing nutrients)

Extends the LAG phase of contaminants and shortens the LAG phase of pathogens
ENRICHMENT
(effective up to 6 hours)
MEDIA

Ex. Alkaline Peptone Water (Vibrio), Selenite broth (gram (-) organisms)
Tetrathionate broth (Salmonella, Shigella), Thioglycollate (anaerobes),
Todd-Hewitt with colistin and nalidixic acid (Group B Strep)
Gram Negative – GN broth (enteric gram (-) organisms)
Clear (No Growth) Turbid (Cloudy) Flocculent Pellicle Sediment

It contains “INHIBITORS” that inhibit the growth of all bacteria GROWTH IN THIOGLYCOLLATE BROTH
except those that are sought.

INHIBITORS FOR GRAM (+) INHIBITORS FOR GRAM (-)

Dyes: Gentian violet, Crystal violet
Potassium tellurite, Sodium azide
Bile salt: Sodium desoxycholate
SELECTIVE EXAMPLES
MEDIA For Gram (+) bacteria Phenylethyl Alcohol (PEA) – inhibits gram (-) bacteria
M. tuberculosis Lowenstein-Kensen (LJ) medium
Petragnani medium – for heavily contaminated specimen Obligate Facultative Aerotolerant
C. diphtheria Potassium Tellurite medium – black colonies Obligate Aerobes Anaerobes Anaerobes Anaerobes Microaerophilic
N. gonorrhoeae Thayer-Martin, Modified Thayer-Martin,
SOD Catalase SOD Catalase SOD Catalase SOD Catalase SOD Catalase
Martin-Lewis, New York City Agar
+ + - - + + + - + +/-
These media allow one to select for pathogens through the inhibition of normal flora

D. Updated Media Classidication based on Function

It provides distinct colonial and biochemical appearances of microorganisms
to aid in their identification
Contain nutrients that support growth of most non-fastidious organisms without gicing
NUTRITIVE any particular organism a growth advantage
Staphylococcus Vibrio
(SUPPORTIVE)
Medium Mannitol Salt agar TCBS agar
DIFFERENTIAL Ex. Trypticase soy agar, Nutrient agar plates, BAP, CAP, SDA
CHO Mannitol Sucrose
(INDICATOR)
Inhibitor 7.5-10% NaCl Bile salts
MEDIA
pH Indicator Phenol Red Bromthymol blue
Contain specific nutrients required for growth of particular bacterial pathogens that may be
Fermenters Yellow colonies Yellow colonies
present alone or with other bacteria
Non-fermenters Pink colonies Green colonies ENRICHMENT
One species or group of bacteria as a certain characteristic that separates it from other Enhances growth of a specific pathogen present in low numbers from a
types of bacteria polymicrobial colony by providing specific nutrients

It maintains moist environment with nutrients for growth and organism viability Contains one or more agents that are INHIBITORY to all organisms except those selected
TRANSPORT
MEDIA by the specific growth condition or chemical
Ex. JEMBEC/TRANSGROW (Neisseria), STUART’S (Viral), CARY BLAIR (Vibrio) SELECTIVE

INHIBITORY AGENTS: dyes, bile salts, alcohols, acids, antibiotics

STOCK CULTURE DIFFERENTIAL Employ some factor/s that allow colonies of one bacterial species to exhibit certain
metabolic or culture characteristics that can be used for distinction
For storage of organisms at -70oC using Schaedler broth with glycerol, chopped meat (anaerobes), skim milk,
tryptic soy agar deeps, and cysteine tryptic agar

Page 31 of 40
 COLONIAL CHARACTERISTICS

TAP WATER AGAR 1. SIZE: large, medium, small, pinpoint

2. FORM (MARGIN): smooth (entire), filamentous, rough, rhizoid, irregular
 Bacto agar is added to 100 mL to tap water, sterilized, and then poured into plates 3. COLOR: white, gray, yellow, buff
 Plates are lightly inoculated using a single streak and incubated at 30oC for up to 7 days and examined daily for 4. ELEVATION: raised, flat, umbilicate (depressed center, concave), umbonate (raised, bulging center, convex)
aerial hyphae 5. DENSITY: transparent, translucent, opaque
 To differentiare aerobic Actinomycetes (Nocardia, Rhodococcus, Gordonia, Tsukamurella, Streptomyces, 6. CONSISTENCY: brittle, creamy, dry, waxy
Actinomadura, Dermatophilus, Nocardiopsis) 7. PIGMENT: green, red, blue, purple, brown, black
8. ODOR
Aerial hyphae (+): Nocardia, Streptomyces, Nocardiopsis
SIZE
QUANTITATIVE STREAK (CROSS-STREAKING)
 Colonies are described as large, medium, small,
or pinpoint
For urine culture and tissues from burn patients using a calibrated loop (1:100 or 1:1000)
 Size is generally a visual comparison between
genera or species.
 gram (+) bacteria generally produce smaller
colonies than gram (-) bacteria
 Staphylococcus species are usually larger
than Streptococcus species

FORM OR MARGIN
Straight line in the middle followed by a zigzag pattern, not overlapping

Colonies counted is multiplied with the calibration used (commonly used is 1:1000, 1:100)  The edge of the colonies are observed and the form (or margin) described as smooth, filamentous, rough or
rhizoid, or irregular
SEMIQUANTITATION (ISOALTION) STREAK

An isolation procedure used for obtaining a pure culture and inoculation in four quadrants

Loops are flamed in between quadrant streaks

MARGIN: smooth (entire), rough, irregular,
Quantification is based on the growth on each quadrant curled, filamentous
1st: 1+ or rare
2nd: 2+ few FORM: observed at the edge of the colony
3rd: 3+ or moderate
4th: 4+ or many SURFACE: dull, glisteing, moist

COLOR

 In contrast to pigmentation, color is a term used to describe a particular genus in general.

 Colonies may be white, gray, yellow, or buff.
 Coagulase-negative staphylococci are white
 Enterococcus spp. may appear gray.
 Certain Micrococcus spp. and Neisseria spp. (non-pathogenic) are yellow or off-white
 Diphtheroids are buff
BUTT SLANT AGAR SLANT AGAR DEEP  Most gram-negative rods are gray on SBA
Stab and Streak Streak Stab

Page 32 of 40
ELEVATION PIGMENT

 The elevation should be determined by tilting the culture plate and looking at the side of the colony  Pigment production is an inherent characteristic of a specific organism confined generally to the colony
 Elevation may be raised, convex, flat, umbilicate (depressed center, concave), or umbonate (raised or o although some pigments will diffuse through the culture medium.
bulging center, convex)  Pigment production is generally enhanced by growing bacteria at room temperature
 S. pneumoniae typically produces umbilicate colonies, unless the colonies are mucoid because of the
presence of a polysaccharide capsule green, sometimes a metallic sheen
 S. aureus typically produces convex colonies. In comparison, β-hemolytic streptococci generally produce flat Pseudomonas aeruginosa
(SBA)
colonies. Not all strains of the same organism have the same colony appearance
brick red (incubation at room temperature enhances pigmentation)
Serratia rubidaea (MAC)
This brick-red pigment should not be confused with lactose fermentation
Kluyvera spp Blue
Chromobacterium violaceum Purple or Violet
Prevotella melaninogenica Brown-black (anaerobic)
Corynebacterium diphtheriae Gun-metal black
Pigment production for these organisms is variable

ODOR

 Odor should be determined when the lid of the culture plate is removed and the odor dissipates into the surrounding
DENSITY
environment.
 The density of the colony can be transparent, translucent, or opaque. The microbiologist should never inhale directly from the plate
 To see the differences in the density of colonies, it is useful to look through the colony while using transillumination.
Translucent colonies allow some light to pass through the colony and opaque colonies do not (Fig. 8.12). BACTERIA ODOR
 Staphylococci and other gram-positive bacteria are usually opaque. Chromobacterium violaceum Almond-like or Ammonium cyanide
o Most gram-negative rods also form opaque colonies. Eikenella corrodens
 β-Hemolytic streptococci except group B (S. agalactiae) are translucent. Bleach-like
Haemophilus influenzae
 S. agalactiae produces colonies that are semiopaque, with the organisms concentrated at the center Proteus spp. Burnt chocolate, Chocolate cake, Gun powder
of the colony, sometimes described as a bull's-eye colony. Beta-hemolytic Streptococci Buttery
 Bordetella pertussis is shiny, similar to a half-pearl, on blood-containing media
Streptococcus anginosus Butter scotch, Sweet honeysuckle
Brevibacterium Cheese-like
Burkholderia cepacia
Dirt-like, Earthy
Burkholderia pseudomallei
Gardnerella vaginalis Fishy amine
Anaerobes Foul
Nocardia spp. Freshly plowed grass or field
Alcaligenes faecalis Fruity apple or strawberries
CONSISTENCY Pseudomonas aerugonisa Fruity (grape-like), Corn tortilla
Clostridium difficile Horse stable, Barn yard
 Consistency is determined by touching the colony with a sterile loop; occasionally, the entire colony adheres (sticks) Stenotrophomonas maltophilia Mousy or Ammonia-like
to the loop Streptomyces Musty basement
 Colony consistency may be brittle (splinters), creamy (butyrous), dry, or waxy Haemophilus spp. Musty basement
 S. aureus is creamy Pasteurella spp. “Mousy” or “Mouse nest”
 Neisseria spp. are sticky Pasteurella multocida Musty, Mushroom
 Nocardia spp. produce colonies that are brittle, crumbly, and wrinkled (resembling bread crumbs on a plate) Serratia odorifera Musty, Pungent, Potato-like
 Diphtheroid colonies are usually dry and waxy Staphylococcus aureus Old sock
 Most β-hemolytic streptococci are dry (except for mucoid types), and when pushed by a loop, the whole
Neisseria zoodegmatis
colony remains intact. Popcorn
Neisseria animaloris
Proteus mirabilis Putrid
Eubacterium
Rancid butter
Fusobacterium nucleatum
Psychrobacter Roses
Peptostreptococcus Sweat, Fetid

Page 33 of 40
GROWTH OF MICROORGANISMS IN LIQUID MEDIA

Important clues to the identification of an organism can also be detected by observing the growth of the organism in
liquid media such as thioglycollate.

A: “Vine” or “streamer” by certain species of Turbidity produced by enterics when growing in

streptococci (more prevalent toward the bottom of the thioglycollate. Notice the gas bubbles at the surface of
tube) and in the middle of the medium (arrow)
B: “Puffed balls” exhibited by certain streptococcal
spp.

Production of “scum” by a yeast at the surface of Pseudomonas sp. producing surface “scum” at the
thioglycollate sides of thioglycollate (P. aeruginosa produces a
diffusible green pigment and a metallic sheen at the
surface)

Yeast in the microaerophilic area of thioglycollate

GROWTH MEASUREMENT

CELL COUNT
Microscopy, Electronic plate counter, Colony count
Petroff-Hausser Counter For microscopic count of prokaryotes
Breed Count Method For microscopic count of bacteria in milk
Most commonly used method; measures the # viable cells (CFU/mL) – 30-
Plate Count
300 colonies – in milk, food, water, and soil
CELL MASS
Weighing (for fungi), measuring nitrogen content, and turbidimetry
CELL ACTIVITY
Relating biochemical activity of bacteria in milk

Page 34 of 40
 ANTISUSCEPTIBILITY TEST (AST) DISK DIFFUSION METHOD “KIRBY-BAUER TEST”

SUSCEPTIBILITY TESTING is done to STANDARD INOCULUM SIZE: 1.5 x 108 CFU/mL

► Detect the ability of antimicrobial agent to inhibit bacterial growth in vitro Principle: Based on the INVERSE linear relationship between the diameter of the zone of inhibited growth around the
► Determine the susceptibility or resistance of organisms against antimicrobial agents antibiotic disk and the logarithm of the MIC of the antibiotic

MINIMUM BACTERICIDAL CONCENTRATION MINIMIMUM INHIBITORY CONCENTRATION (MIC)  Limited to aerobic and facultatively anaerobic bacteria
(MBC) MINIMUM LEHTAL CONCENTRATION (MLC)  Filter paper disks impregnated with various antimicrobial agents of specific concentrations are carefully placed on
The lowest antibiotic concentration that The lowest antibiotic concentration that an agar plate previously inoculated with the bacterium being tested
killed the bacterium yielding 99.9% reduction inhibits in vitro visible growth  Kirby-Bauer test: routinely used to determine the S/R of a pathogenic organism to various antimicrobial agents
TOLERANCE  Susceptibility Standard Medium: Mueller Hinton Agar (MHA)
If an antibiotic’s MBC is ≥ 32 times higher than the MIC, the organism may be tolerant to the drug  MIC is NOT reported; it is impossible to quantify the amount of antimicrobial agent diffused into the agar medium
 After incubation, diameter of the zone of inhibition around each disk is measured in millimeters (mm)
a phenomenon most commonly associated with bacterial resistance to beta-lactam antibiotics; o Results are reported as: SUSCEPTIBLE (S), INTERMEDIATE (I), RESISTANT (R)
reflects an organism’s ability to be inhibited by an agent that is sually bactericidal o The more RESISTANT bacterial strains, the corresponding inhibition zone sizes DECREASES
Direct measures of antimicrobial activity are accomplished using:
1. Convevntional suscepbility testing methods such as: broth dilution, agar dilution, disk diffusion MIC have an INVERSE relationship with ZOI
2. Commercial susceptibility testing systems
3. Special screens and indicator tests
PREPARATION OF CULTURE FOR SUSCEPTIBILITY TESTING
TEST TUBE DILUTION “BROTH DILUTION METHOD”
VARIABLE STANDARDS NOTE
1.5 x 108 Use adequate McFarland turbidity standard
STANDARD INOCULUM SIZE: 5 x 105 CFU/mL Inoculum
CFU/mL (0.5% for disk diffusion)
Prepare in house or purchase from reliable source.
 Involves challenging the organism of interest with antimicrobial agents in a broth environment (Mueller-Hinton broth) Formulation Mueller-Hinton Perform media quality control ro verify acceptability before
o Each antimicrobial agent is tested using a range of concentrations, commonly expressed as micrograms use for patient tests
(ug) of active drug per milliliter (mL) of broth “ug/mL” Increased (↑) Concentration
 A specific amount of antibiotic is prepared in a decreasing concentration in broth by serial dilution techniques, and Calcium 25 mg/mL
standard numbers of the test organism is inoculated ► (↓) activity of Aminoglycosides against P. aeruginosa
and ↓ activity of Tetracyclines against all organism
 Concentration range examined for a particular drug depends on specific criteria, including the safest therapeutic ► (↑) RESISTANCE of [Link] to
Magnesium 12.5 mg/mL
concentration possible in a patient’s serum

Media
Aminoglycosides * False Resistance
 Can be used to determine MIC and MBC Minimal or Escessive concentrations can result in FALSE
Thymidine
Absent RESISTANCE to Sulfonamides and Trimethoprim
TYPES OF BROTH MICRODILUTION METHOD acidic pH
MICRODILUTION MACRODILUTION
pH 7.2-7.4 ► (↓) activity of “ACE” Aminoglycosides, Erythromycin,
 Various concentrations of several antibiotics are  The MIC and MBC are determined through and Clindamycin ∴ FALSE RESISTANT
tested against an inoculum serially diluting an antibiotic ► (↑) activity of Tetracyclines ∴ FALSE SUSCEPTIBLE
 The MIC is determined using this method  Useful for laboratories testing a few antibiotics Agar Depth 3-5 mm <3 mm: FALSE SUSCEPTIBILITY (no space for growth)
 It uses MULTIWELL MICRODILUTION TRAYS with a few organisms (Disk Diffusion) (average 4 mm) >5 mm: FALSE RESISTANT (bacteria burrows)
 Broth Volume: 0.05-0.1 mL  It uses TEST TUBE CO2 incubation decreases (↓) pH leading to:

Incubatuion
 Schaedler’s broth  The REFERENCE METHOD FOR ANTIBIOTIC Humidified
Atmosphere ► (↓) activity of Aminoglycosides, Erythromycin, and
 West Wilkin’s broth SUSCEPTIBIITY TESTING Ambient Air
Clindamycin
 Brain Heart Infusion (BHI)  Broth Volume: >10 mL ► (↑) activity of Tetracyclines
Temperature 35o ± 2 oC Some MRSA may go undetected if >35 oC
AGAR DILUTION METHOD Length 16-24 hours Some MRSA may go undetected if <24 hours
Long Term: non-frost-free freezer at ≤ -20 oC in tightly
STANDARD INOCULUM SIZE: 1 x 104 CFU/mL sealed, dessicated container

Antimicrobial Agents Short Term (at least 1 week): maintain 2-8 oC in tightly
 The antimicrobial concentrations and organisms to be tested are brought together on an agar-based medium (Disks)
Proper Storage
sealed, dessicated container
 Diffusion of the drug in the agar establishes a concentration gradient from high (center of plate) to loe (periphery of
plate) Allow to warm to room temperature before opening
 The shelf-life of an agar dilution plate is only 1 week for most antimicrobial agents container
 The REFERENCE METHOD for testing Anaerobes and N. gononrrhoeae: Water
BEST: deionized water
(for media preparation)
► ANAEROBES: Brucella agar with laked blood and Vitamin K (Wadsworth Method)

Page 35 of 40
 0.5 MCFARLAND STANDARD 99.5 mL of 1% H2SO4 + 0.5 mL of 1.175% BaCl IN CASE OF FASTIDIOUS ORGANISMS
ANTIBIOTIC DISK SIZE 6 mm
STREAKING METHOD Overlap streaking using cotton swab Streptococcus pneumoniae MHA with 5% sRBCs
DISTANCE OF THE DISK FROM THE CENTER 24 mm Haemophilus Test Medium (HTM); MHA with hematin (X), NAD (V), and
DISTANCE BETWEEN TWO DISKS 15 mm Haemophilus influenzae
yeast extract
(35o-37 oC) for 16-18 hours
INCUBATION TIME AND TEMPERATURE MHA with 2% NaCl incubated for 24 hours at 30-35oC
Some MRSA may go undetected if >35oC MRSA
ZONE OF INHIBITION MEASUREMENT Ruler or Caliper Does not grow <24 hours and >35 oC
# OF ANTIBIOTIC DISKS (150 mm plate) No more than 12 disks Neisseria meningitidis MHA with 2-5% lysed horse blood
# OF ANTIBIOTIC DISKS (100 mm plate) No more than 5 disks Neisseria gonorrhoeae GC agar with supplements
HIGHEST CONCENTRATION Closest to the disk - CENTER Mycobacteria Middlebrook 7H10
Frozen in non-frost-free freezer at ≤ -20oC in ightly Anaerobes
LONG-TERM STORAGE Brucella blood agar with hemin incubated anaerobically up to 48 hours
sealed, dessicated container
SHORT-TERM STORAGE 2-8 oC at least 1 week
Plates should be inverted (to prevent moist) and not stacked more than five plates when incubating MHA + 5% sRBC and HTM are incubated in 5-7% CO2 for 18-20 hours
AST for Streptococci (Fasitidious): MHA + Blood

1. Subculture 4-5 colonies on TSB from pure culture and incubated at 35oC for 3-5 hours (mid-logarithmic
phase) REMEMBER
2. Compare turbidity of subculture with McFarland standard (Purpose: to standardize the inoculum)
3. If turbidity is present, inoculate (lawning – streaking the agar in all directions) on MHA ► INDICATOR OF IMPROPER STORAGE: Methicillin, Penicillin (antibiotics first to degrade)
(too much turbidity = dilution with NSS or distilled water) ► ACIDIC pH affects Tetracycline, Novobiocin, Methiliccin
4. Wait for 3-5 minutes (NOT longer than 15 minutes) before applying the disks ► ALKALINE pH affects Aminoglycosides, Clindamycin, Erythromycin
► Swarming on Kirby Bauer (MHA) is IGNORED, zones are measured at the point where growth is
5. Apply antibiotic disks 24mm from center to center
obviously inhited however, it is considered significant on BAP
6. Invert plate and incubate 35-37oC for 16-24 hours ► Susceptibility to Sulfonamdes have two (2) concentric rings around the disk, measure the outer zone
7. Measurement of the zone of inhibition is aided by a ruler or caliper with naked eye, interpreted as S, R, ► Hazes of bacterial growth may be observed when testing Sulfonamide and Trimethoprim (SXT) as
or I (should be underside, for media with blood, measure from the top with cover removed) a result of the organism population going through several doubling generations before inhibition
» The resulting haze of growth should be IGNORED for disk interpretation with these agents
Zone of Inhibition (ZOI) is interpreted using a dark background and reflected light ► Zone diameters are too large or too small with all antimicrobials
ZOI  Check inoculum size (most important)
Reflected light
HEMOLYSIS Transmitted light  Check the agar depth, if incorrect, adjust the amount of agar dispensed
 Ensure that disks are applied to inoculated plates within 15 mnutes of application of disks
► Once the disk is placed on the inoculated plate and makes contact with the agar, it begins to diffuse out;
Visible growth inside the zone of inhibition indicates inoculation with a mixed culture however, emergence of the concentration of the antiobiotic gets lovwer the further it diffuses from the disk
resistant mutants of test isolate is a more likely reason for this growth pattern (ESBL, MRSA, VRE)

FACTORS AFFECTING THE ZONE OF INHIBITION

FALSE RESISTANT FALSE SUSCEPTIBLE QUALITY CONTROL SATISFACTORY FROM DAILY TO WEEKLY
(Smaller zone) (Larger zone)
 Increase moisture  Increase drying (dry agar) ► Susceptibility testing of control organisms is usually conducted daily until precision can be demonstrated
 Heavy inoculum  Light inoculum wth 20-30 consecutive days of susceptibility testing
 Thick medium: antibiotic cannot diffuse into agar  Thin medium ► Once the 20- or 30- day evaluation has been accomplished, quality control organism may be tested
 Prolonged incubation  Delay before incubation weekly instead of daily
 Delay before disc application  Low temperature and old colonies
 Use of mixed culture
 Reading and clerical error
 Deterioration of turbidity standard or control strains

Page 36 of 40
D TEST INTRAVENOUS INTRAMUSCULAR ORAL
30-60 minutes 60 minutes 90 minutes
 To detect inducible clindamycin resistance among strains of S. aureus demonstrating an initial antibiotic
susceptibility profile (initial result is DISCREPANT):  The SBT was originally developed to aid in predicting the clinical efficacy of antimicrobial therapy for staphylococcal
► Clnidamycin (Susceptible) endocarditis
► Erythromycin (Resistant)
MODIFIED HODGE TEST
Both antibiotics should have proportional results; Erythromycin induces the clindamycin resistance
Principle: Carbapenemase production is detected by examining a test isolate for the production of the enzyme, allowing
 Uses 15 ug Erythromycin and 2 ug Clindamycin which are positioned 15-20 mm apart growth of a carbapenem-susceptible organism to grow toward the carbapenem disk. The resulting growth
pattern appears as a cloverleaf-like indention.
(+): blunting or flattening of Clindamycin zone to produce a “D” pattern
(-): no flattening (only erythromycin is resistant)  Detect the ability of the organism (Enterobacteriaceae) to produce the enzyme CARBAPENEMASE causing
resistance to Carbapenem (Beta-lactam)

clover leaf-like pattern of zone of inhibition; indented growth of Escherichia coli

(+): control strain toward the carbapenem disk
(+) D-test: Clindamyin = RESISTANT
(-) D-test: Clindamycin = SENSITIVE (-): No indentation of growth of the E. coli control strain from the zone of inhibition
ERM GENE: the gene that activates
resistance to clindamycin

Quality Control: E. coli ATCC 25922

carbapenem susceptible
E “EPSILOMETER” TEST

 A dilution method that uses thin plastic test strips impregnated on the undersurface with an antimicrobial
concentration gradient (varying concentration) and marked on the upper surface with a concentration index or scale
 NOT ROUTINELY USED BUT FOR RESEARCH PURPOSES (due to its cost) SUPPLEMENTAL METHODS FOR DETECTION OF ANTIMICROBIAL RESISTANCE
 Suited for fastidious organisms and anaerobes
 Has an advantage due to the number of antibiotics in the strip equipped with different concentration
OXACILLIN AGAR Detection of STAPHYLOCOCCAL RESISTANCE to Penicillinase-Resistance
 Antimicrobial gradient forms in the agar around the E-test strips giving rise to elliptic inhibitory areas with each
strip SCREEN Penicillins (e.g., Oxacillin, Methicillin, Nafcillin)
 MIC is determined where the growth inhibition edge “ellipse” intersects the E-test strips OXACILLIN DISK SCREEN Differentiate Clindamycin Resistance among S. aureus resulting from efflux
VANCOMYCIN AGAR
Detection of ENTEROCOCCAL RESISTANCE to Vancomycin
SCREEN
AMINOGLYCOSIDE Detection of acquired enterococcal high-level resistance among Aminoglycosides that
SCREEN would compromise synergy with a cell wall-active agent (e.g., Ampicllin, Vancomycin)
(+): ellipse of growth inhibition

SERUM BACTERICIDAL TEST “SCHLICTER TEST”

 Done to determine if the patient is receiving effective treatment for infection

 Patient’s serum (test medium) containing the therapeutic antimicrobial agents the patient has been receiving
o Using the patient’s serum to detect bacteriostatic and bactericidal activity allows observation of the
antibacterial effect of factors other than the antibiotics (e.g., antibodies and complement)

SAMPLES
1. One is collected just before (within 30 minutes) the patient is to receive the next antimicrobial dose (i.e.,
trough specimen)
2. Collected after the antimicrobial agent(s) is given when the serum antimicrobial concentration is highest
(i.e., peak specimen)

 The appropriate time to collect the peak specimen varies with pharmacokinetic properties of the antimicrobial agents
and their route of administration
Page 37 of 40
AUTOMATED SYSTEM

ADVANTAGES: High sensitivity, Reduce TAT, Replaces manual procedure

Principles of Identification

Commercial identification systems fall into one of five categories or a combination thereof:
(1) pH-based reactions
(2) enzyme-based reactions,
(3) utilization of carbon sources
(4) visual detection of bacterial growth
(5) detection of volatile or nonvolatile fatty acids by gas chromatography

 64-well, closed card containing specified concentrtions of antibiotics

 MIC results are validated with the Advanced Expert System (AES) Software, a
VITEK LEGACY and
category interpretation is assigned and the organism’s antimicrobial resistance
VITEK 2 SYSTEM
patterns are reported
 Monitors the Optical Density (OD) reading
 Uses broth microdilution panel format that is manually inoculated with a
WALK-AWAY SYSTEM multiprong device
 Bacterial growth may be detected spectrophotometrically or fluorometrically
 Manual, gravity-based inoculation process
PHOENIX SYSTEM  Growth is monitored in an automated fashion based on a redox indicator system
 Interpretation is augmented by a rules-based data management expert sytem

COMMERCIALLY AVAILABLE AUTOMATED SYSTEMS FOR MICROBIAL IDENTIFICATION

NAME MANUFACTURER PRINCIPLE ORGANISMS IDENTIFIED
Carbohydrate utilization  Enterobacteriaceae
BD Diagnostic
BD Phoenix Chromogenic substrate  other GN bacilli
Systems
Fluorogenic substrate  GP
 Enterobacteriaceae
 other GN bacilli
 Neisseria spp
 Haemophilus spp
Carbohydrate utilization
MicroScan  Streptococci
Beckman Coulter Chromogenic substrate
(Autoscan, WalkAway)  Enterococci
Fluorogenic substrate
 Staphylococci
 GP bacilli
 Yeasts
 Anaerobes
 GN organisms
Carbohydrate utilization
OmniLog Biolog  GP organisms
Tetrazolium violet reduction
 Anaerobes
 GN organisms
ThermoFisher Carbohydrate utilization
Sensititire (TREK)  GP organisms
Scientific Chromogenic substrate
 Anaerobes
 GN organisms
Sherlock Microbial Fatty acid analysis of microbial  GP organisms,
MIDI
Identification System cells  Mycobacterium spp.
 Yeasts
 Enterobacteriaceae
 other GN bacilli
 Neisseria spp
 Haemophilus spp
Carbohydrate utilization  Streptococci
Vitek 2 bioMérieux
Chromogenic substrate  Enterococci
 Staphylococci
 GP bacilli
 Yeasts
 Anaerobes

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 ANTIBIOTICS MODE OF ACTION CLASS REPRESENTATIVE ANTIBIOTICS
Tetracyclin
BROAD SPECTRUM NARROW SPECTRUM Doxycycline
Tetracycline
Minocyclin
Destroys different kinds of organisms Effective against a limited number of organisms (inhibits protein synthesis at 30S RSU)
Oxytetracycline
Demeclocycline
BACTERIOSTATIC BACTERICIDAL
Kanamycin
Inhibit the microorganism Destroy or kill the organism Aminoglycoside Gentamycin
o
STORAGE TEMPERATURE FOR ANTIBIOTIC DISKS (Working Supply): 2-8 C (inhibits protein synthesis at 30S RSU) Amikacin
PROTEIN
Tobramycin
MODE OF ACTION CLASS REPRESENTATIVE ANTIBIOTICS SYNTHESIS
Erythromycin
Colistin INHIBITORS Macrolide
Clarithomycin
Nystatin (inhibits protein synthesis at 50S RSU)
CELL MEMBRANE Azithromycin
Polymyxins Amphotericin B Dirithromycin
INHIBITORS Lincosamie
Polymyxin Lincomycin
Bacitracin Streptogramin
Lindamycin
*Antibiotic of last resort; long term
MODE OF ACTION CLASS REPRESENTATIVE ANTIBIOTICS Chloramphenicol
use may cause bone marrow
Penicillin V (inhibits protein synthesis at 50S RSU)
Natural Penicillin from damage and lead to aplastic anemia
Penicillin notatum
Penicillin G  Tetracycline
Benzathine penicillin o BROASDEST spectrum: gram +/-, atypical; and intracellular
Methicillin o Not advisable for children as it inhibits bone growth
Oxacillin
Synthetic Pencillin Carbenicillin  Doxycycline (200mg) for Leptospirosis prevention
BETA-LACTAM

Ampicillin o taken within 24-72 hours intended for those in flooded areas
Piperacillin o once a day for 3-5 days for wounded individual in flooded area
1st: Cephalexin
CELL WALL 2nd: Cefoxitin mycin: obtained from Streptomyces
Cephalosporin NOTES
INHIBITORS 3rd: Ceftriaxone, Cefixime micin: obtained from Micromonospora
4th: Cefepime  Aminoglycodes
Imipenem o AUTOTOXIC and NEPHROTOXIC
Carbapenem Meropenem o More active on gram (-) and some gram (+)
Ertapenem
 Erythromycin, now CREDE’S PROPHYLAXIS for opthalmia neonatorium prevention
Aztreonams
Monobactam  Chloramphenicol
Tigemonam
Vancomycin o BROASD spectrum for serious gram (-) infections (e.g, meningitis)
Glycopeptide o TOXIC TO BONE MARROW causing aplastic anemia
Teicoplanin
Cyclic peptide Bactracin
 BETA-LACTAM inhibits TRANSPEPTIDASE in cell wall MODE OF ACTION CLASS REPRESENTATIVE ANTIBIOTICS
 Organisms with beta-lactamase are RESISTANT to beta-lactams FOLIC ACID
Sulfonamides Sulfamethoxazole
 Extended Spectrum Beta Lactamase (ESBL): RESISTANT to 3rd generation SYNTHESIS
(outer ZOI is measured) Trimethoprim
cephalosporins INHIBITORS
o ESBL (+) treatment is CARBAPENEMS Sulfonamides prevent purine production (precursor of DNA)
o ESBL (+): E. coli, Klebsiella SULFANILAMIDE is an antimicrobial agent BUT NOT AN ANTIBIOTIC; it is not
produced by an organism
NOTES
Resistance to beta-lactams leads to shift in drug utilization;  Sulfonamide: inhibits dihhydropteroate synthase
use of anti-beta-lactamase drugs or use of higher generation of drugs  Trimethoprim: inhibits dihydrofolate reductase
 Cotrimoxazole: common name of SXT
NOTES
K. pneumoniae Carbapenemase (KPC) gene (+) shows resistance to Carbapenems
MODE OF ACTION CLASS REPRESENTATIVE ANTIBIOTICS
RESISTANT TO Carbapenem: KPC gene (+) Rifampin
RESISTANT TO 3rs Gen: ESBL (+) (inhibits RNA polymerase beta-subunit)
DNA
 Anti-beta-lactamase: CLAVULANIC ACID, SULBACTAM, TAZOBACTAM Metranidazole
(NUCLEIC ACID)
 Vancomycin (disruption of DNA)
SYNTHESIS
o drug of choice for MRSA INHIBITORS Ciprofloxacin
Quinolones
o NEPHROTOXIC causing “Red Man Syndrome” Ofloxacin
(binds to DNA gyrase)
 Bacitracin (aka Taxo A): 0.04 u for S. pyogenes Levofloxacin
 Rifampicin
o Advised for TB patients
NOTES o produces orange-red urine and other body fluids
 Quinolones: binds to DNA gyrase

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 CATEGORIZATION OF BACTERIA ACCORDING TO NEED FOR ROUTINE PERFORMANCE OF AST
Testing Commonly Required Testing Occasionaly Required Testing Rarely Required
Staphylococci Haemophilus influenza B-hemolytic streptococci (groups A, B, C, F, G)
Streptococcus pneumonia Neisseria gonorrhee Neissieria meningitidis
Viridans streptococci Moraxella catarrhalis Listeria monocytogenes
Enterococci Anaerobic bacteria
Enterobacteriaceae
Pseudomonas aeruginosa
Acinetobacter spp.

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