MICROBIOLOGY (REVIEW) BACTERIAL CYTOLOGY = 70% WATER, 30% CHO AND

SPEARHEAD CAPITOL REVIEW CENTER CHONS, LIPIDS ETC

IMPORTANT TOPICS:
 Introduction to Bacteriology
 Important Parts of Bacterial Cell
 Bacterial Physiology / Requirements for Bacterial Growth
 Stages of Bacterial Growth
 Concepts on Laboratory Biosafety
 Biosafety Cabinet
 Methods of Microbial Control
 Collection Transport & Specimen Processing
 Specimen Storage
 Clinical Specimen
 Bacterial Identification
 Susceptibility Testing
 Antimicrobial Agent
 Gram stains:
o Gram Positive Cocci
o Gram Negative Cocci

INTRODUCTION TO BACTERIOLOGY Exotoxin Endotoxin

 MICROBIOLOGY – Microbiology is the study of the
Species Some species of Most Gram-negative
biology of microscopic organisms - viruses, bacteria,
Gram-positive and bacteria and Listeria
algae, fungi, slime molds, and protozoa. The methods
Gram-negative
used to study and manipulate these minute and mostly
bacteria
unicellular organisms differ from those used in most other
Protein _________________ Part of cell
biological investigations (University of Pittsburg)
Location (lipopolysaccharide)
 BACTERIOLOGY – The science and study of bacteria and that fragments off
their relation to medicine and to other areas such as Genes for exotoxin Genes for endotoxin
Gene
agriculture (e.g., farm animals) and industry. are in plasmid or are on bacterial
Location
bacteriophage chromosome
IMPORTANT CHARACTERISTICS OF BACTERIA Toxicity _________________ Low toxicity
 Prokaryote – No true nucleus
Antigenicity Highly antigenic (host Poorly antigenic
 Has both RNA and DNA; Multiplies via binary fission.
forms antibodies
 Measured in um = average size = 0.4 to 2 m. called antitoxins)
 Virulence factor = Produce either endotoxin or exotoxin Vaccine Vaccine available _________________
(formed from toxoids)
1
 Heat Heat labile Heat stable Lipotechoic acid Present Absent
Stability Lipoproteins Absent Present
Example Think cholera, Think Lipopolysaccaharide
Absent Present
tetanus, botulism meningococcemia, layer
sepsis

IMPORTANT PARTS OF BACTERIAL CELL

1. CELL WALL - Defines the shape of the bacteria, the main
component is peptidoglycan. A.k.a ______________
________________
 Point of anchorage for flagella.
 Site of antibiotic action
 Basis of gram staining gram positive with rigid
peptidoglycan layer while gram negative with thinner
peptidoglycan layer; determines gram positivity/gram
negativity of an organism.
Gram (+) cell wall (impermeable to alcohol) with teichoic acid and
N-Acetyl Glucosamine and N-Actyl Muramic acid GRAM STAINING
Gram (-) cell wall: (permeable to alcohol) with CHON,
 Discovered by "Hans Christian Gram' to distinguish and
phospholipids & LPS -> no teichoic acid
differentiate gram positive and gram-negative
Mycoplasma and Ureaplasma No cell wall
microorganism.
 Gram Positive: with thick peptidoglycan layer.
2. _____________– Slimy area surrounding the cell wall;
 Gram Negative: with thin peptidoglycan layer.
Function is “anti-phagocytic”.
 Bacillus anthracis
 Klebesiella pneumoniae
 Haemophilus influenzae
 Steprococcus pneumoniae

Neufeld Quellung – Capsular swelling test

CELL WALL GRAM

GRAM POSITIVE
COMPONENT NEGATIVE
Peptidoglycan Thick layer Thin layer
IMPORTANT NOTES:
Periplasmic
Absent Present  ALL COCCI ARE GRAM POSITIVE EXCEPT:
space/layer
“NeVer Mind the Boys”
Outer Membrane Absent Present
o N -eiserria
Teichoic acid Present Absent
2
 o V -eilonella o Bacillus
o M -oraxella o Erysipelothrix
o B -ranhamella o Lactobacillus
o Listeria
 ALL BACILLI ARE GRAM NEGATIVE EXCEPT
“CALL BC MEN”  GRAM NEGATIVE BACILLI (AEROBES)
o C -lostridum o Acinetobacter
o A -actinomyces o Alcaligens
o L -actobacillus o Brucella
o L -isteria o Francisella
o B -acillus o Pasteurella
o C -ornybacterium o Vibrio
o M -ycobacterium o Aeromonas
o E -rysiphelothrix o Bordetella
o N -ocardia o Enterobacteriaceae
o Legionella
 GRAM POSITIVE COCCI (AEROBES) o Pseudomonas
o Staphylococcus
o Streptococcus  GRAM POSITIVE BACILLI (ANAEROBES)
o Micrococcus o Clostridium
o Actinomyces
 GRAM NEGATIVE COCCI (AEROBES) o Propionibacterium / Cutibacterium
o Branhamella
o Neisseria  GRAM NEGATIVE BACILLI (ANAEROBES)
o Fusobacterium
 GRAM POSITIVE COCCI (ANAEROBES) o Bacteroides
o Peptococcus
o Peptostreptococcus 3. _______________________
o Sarcina • Surrounds the cytoplasm; site of energy synthesis
transport of nutrients in and out of the cell; Function
 GRAM NEGATIVE COCCI (ANAEROBES) as golgi apparatus & lysosome.
o Vielonella 4. PILI OR FIMBRIAE
• usually found in gram negative bacteria like E. coli,
 GRAM POSITIVE BACILLI (AEROBES) N. gonorrhoeae, Pseudomonas
o Nocardia
COMMON PILI – For adherence or attachment to host
o Mycobacterium
cells.
o Corynebacterium
SEX PILI – For genetic Conjugation
3
5. ENDOSPORES 2. Flagellar staining – Gray’s and Leifson’s
• Resistant structure enabling bacteria to withstand 3. Use of Semi solid media –SIM
adverse conditions, Target of sterilization. Classification Of Bacteria According to Flagella
• The best way to destroy spores is by  ________________– with single flagellum at one end
AUTOCLAVING. i.e., Vibrio and P. aeruginosa
• Resistant because of CALCIUM DIPICOLINATE /  AMPHITRICHOUS - single flagellum at both ends i.e.,
DIPICOLINIC ACID C. jejuni
• Bacteria with spores  ATRICHOUS- absence of flagella
C. tetani – terminal end. • C. botulinum – subterminal  LOPHOTRICHOUS - tuft of flagella at one or both ends
B. anthracis - central i.e., S. maltophilia
 PERITRICHOUS – surrounded with flagella i.e., [Link]

 GLIDING motility = _____________

 DARTING motility = Campylobacter
 TUMBLING motility = Listeria
 TWITCHING motility = Kingella kingae
 Shooting star motility= ____________
 Cork screw motility = Spirochetes

7. _____________________________ - serves as energy

source; these are food reserves.
 Corynebacterium diphtheriae – Babes ernst
granules
*Yersinia pestis
 Mycobacterium tuberculosis – Much granules
6. FLAGELLA
*Nocardia & Actinomycetes
• For locomotion
8. Nucleoid - The DNA in the bacterial cell is generally
• Manybacilli and spiral are motile while true motility
confined to this central region.
is seldom observed in cocci.
9. Ribosome - Protein synthesis; Gives the cytoplasm of
• Motility best seen at Room temperature – 25
bacteria a granular appearance in EM.
degrees Celsius.
• True motility is due to the presence of flagella.
PLASMID – extrachromosomal DNA carries antibiotic
• Brownian movement of non- motile organisms due
resistant gene.
to the movement of molecules surrounding them.
 Small – Tetracycline and Chloramphenicol
• Microscopes use to visualize spirochetes (1)
Fluorescence Microscope & Dark Field Microscope  Large – Beta lactams
Ways to Demonstrate Motility
1. Hanging drop
4
 III. Temperature
 ____________– cold loving bacteria (0-20 degC)
 Mesophilic – Moderate temperature (20-45 degC)
 ____________– Heat loving organism (20-125 degC)
 Thermoduric – bacteria which do not usually grow at
high temperature but can withstand exposure to high
temperature.

Optimum temperature – defined as the temperature at which an

organism grows best; temperature best suited for bacterial
growth.
COLD ENRICHMENT – is incubation at 4 degC at several weeks
ex. Yersinia interocolitica and Listeria spp.
Bacillus stearothermophilus – thermophilic – Biological
BACTERIAL PHYSIOLOGY / REQUIREMENTS FOR indicator of autoclave.
BACTERIAL GROWTH Thermus aquaticus – DNA Polymerase used in PCR testing.
I. Nutritional Requirements  Incubation temp for most bacteria and viruses – _____
 Phototrophic – uses light as energy source.
 Incubation temp for fungi – RT – 27-30°C
 Chemotrophic – uses chemical compounds.
 Incubation temp and time for AEROBE – 37°C for 18-24
o Autotrophs/lithotrops – uses INORGANIC
COMPOUNDS as carbon source ex. CO2. hrs.
o Heterotrophs/ Organotrophs – uses ORGANIC  Incubation temp and time for ANAEROBE - 37°C for 28-
COMPOUNDS as carbon source ex. 48°C

II. Oxygen Requirement ANAEROBIC CULTURE –(Gas Pak Jar)

 Obligate aerobe – Organisms requiring oxygen for ________________________________
growth ex. Brucella spp., Francisella, Mycobacterium MICROAEROPHILIC – (Candle Jar)
 Obligate anerobe – Organisms NOT requiring oxygen 5% CO2, 10% H2, 85% N2
for growth ex. Veilonella, Actinomyces
 Facultative anaerobe – Organisms that can live with or IV. pH Requirement
without air ex. Family enterobacteriacea o Acidophilic – Requiring acid medium for growth ex.
 Microaerophilic – Organisms requiring SMALL amount lactobacillus.
of air ex. Campylobacter spp. o Basophilic – Requiring alkaline medium for growth
ex. Vibrio.
 Aerotolerant – Does not grow well but survives in the o Neutrophilic – Those able to grow in medium with a
presence of air. Ex. Lactobacillus pH between 7.2 – 7.4
*Extremophiles – able to survive on unusual conditions ex. pH media for fungi – _____________
Absence of O2 and increased temperature. Ex. Bacillus infernus
5
 V. Moisture – 70% moisture is needed to prevent drying,
those requiring increased moisture – HUMIDOPHILIC. LABORATORY BIOSAFETY
VI. _______________ – Those requiring salt concentration  Containment principles, technologies, and practices
are called HALOPHILIC ex. Vibrio spp. implemented to prevent unintentional exposure to
6.5 % - Enterococci spp pathogens and toxins, or their unintentional release.
8 – 10% - Vibrio spp.
7.5% - ______________ PROTECTING PEOPLE FROM DANGEROUS PATHOGENS
VII. Carbon Dioxide ______________– Example: Neiserria LABORATORY BIOSECURITY
spp. and Haemophilus influenzae  Institutional and personal security measures designed to
prevent the loss, theft, misuse, diversion, or intentional
STAGES OF BACTERIAL GROWTH release of pathogens and toxins.

PROTECTING PATHOGENS FROM DANGEROUS PEOPLE.

LABORATORY BIOSAFETY
 Containment principles, technologies, and practices
implemented to prevent unintentional exposure to
pathogens and toxin or their accidental release.
o Protect users.
o Protect those outside the labs.
o Protect the environment.

1. _______________ – No increase in number of living

bacterial cells.
2. _______________ – Exponential increase in number of
living bacterial cells.
3. STATIONARY PHASE – Plateau in number of living
bacterial cells; rate if cell division and death roughly equal.
4. DEATH OR DECLINE PHASE – exponential decrease in
number of living bacterial cells.

CONCEPTS ON LABORATORY BIOSAFETY

According to WHO:

The risk associated with biological materials in the laboratory has

a safety and a security component.
6
 BIOSAFETY CABINET CLASS III
o Most effective system that is entirely closed.
CLASS I CLASS II o Infectious material handled using gloves.
o Partly open and partly
o System is entirely close system
open. o Sterilizes the air
o Negative pressure, entering and circulating
ventilated cabinet, within the cabinet &
Unsterilized air enters exhausts the air.
and circulates within o Sterilizes air to be
the cabinet & the exhausted and the air
exhaust air is filtered that flows over
by HEPA. infectious material. RECALL QUESTION
o AKA “laminar flow” 1. What type of biological safety cabinet is seen in the photo?
a. BSC Class 1
o Process bacterial and b. BSC Class 2
Fungal c. BSC Class 3
o Sterilizes air only to be
Pathogens/Medium d. BSC Class 4
exhausted.
Risk Oncogenic virus/
o Processes NON-
Chemicals/carcinogens
PATHOGENS/BSL 1
.
o Low oncogenic
o IIA – With fixed opening
Virus/Low Toxic
– 70% air circulated.
chemical/Carcinogens
o IIB – Variable sash
.
opening – Exhaust air is
o With 1 HEPA Filter
discharged outside the
bldg.

METHODS OF MICROBIAL CONTROL

 Sterilization - refers to the destruction of all forms of life,
including bacterial spores. By definition, there are no
degrees of sterilization—it is an all-or-nothing process.
7
 Chemical or physical methods may be used to accomplish o Can remove objects larger than 0.3 um.
this form of microbial destruction.
 Disinfection refers to a process that eliminates a defined IV. IONIZING RADIATION/COLD STERILIZATION -
scope of microorganisms. Physical or chemical methods This method of sterilization is used by the medical
may be used. Disinfectants are chemical agents applied to field for the sterilization of disposable supplies such
inanimate objects to kill or destroy disease-producing as syringes, catheters, and gloves.
microorganisms.
2. CHEMICAL METHOD OF STERILIZATION
A. STERILIZATION A. Ethylene oxide (gas sterilant) – most commonly used
1. PHYSICAL METHODS OF STERILIZATION chemical sterilant, used in gaseous form of sterilizing heat
I. ___________________ sensitive object; disinfectant for machine that cannot be
____________– 121°C, 15 psi, 15-30 mins – autoclaved or heated.
Sterilized used and unused media. INDICATOR: Bacillus subtilis var globijii
- 132°C, 15 psi, 30 – 60 mins – Sterilized B. Formaldehyde vapor and vapor phase hydrogen
medical waste. peroxide - used to sterilize HEPA filters in BSCs.
INDICATOR: ________________________ C. Glutaraldehyde (2%) sporicidal, kills spores in 3-10
TYNDALLIZATION – uses flowing steam; equipment used hours, for medical equipment.
in Arnold Sterilizer; 100°C, for 30 mins for 3 consecutive days. D. Peracetic acid
INSPISSATION – thickening through evaporation; For
sterilizing media with increase CHON like Lowenstein Media – for B. DISINFECTION
MTB. 1. PHYSICAL METHODS OF DISINFECTION
II. DRY HEAT - may also be used as a sterilizing I. PASTEURIZATION - _________________ (Low
agent, although it requires much longer exposure temperature Holding) – Remove pathogens in food;
times and higher temperatures than moist heat (1.5 Vat Pasteurization; Batch method.
to 3 hours at 160 – 1800C). This method may be o 72°C, 15 secs (High temperature, short time)
used for heat-stable substances that are not – flash method
penetrated by moist heat, such as oils. Dry heat o 140°C 3 secs (Ultra High temperature, short
oven is commonly used to sterilize glassware. time)

III. ______________ – If can’t be heated, for II. BOILING – 100°C, 15- 30 mins – For surgical
antibiotic solutions, toxic chemicals. instruments
o consist of granular material i.e., berkefield filter, III. UV – non-ionizing radiation using UV light.
asbestos
o these are porous membranes 0.1 mm thick to 2. CHEMICAL METHODS OF DISINFECTION
sterilize culture media, antibiotics. I. Antiseptic – chemical germicide for use on the skin
o Composed of cellulose acetate and polycarbonate or tissue and not to be substituted for a disinfectant.
o Millipore filter with a pore diameter of 22 u can give o Alcohol – most commonly used; Ethyl or
100% sterility isopropyl alcohol (nonsporicidal)
8
 o Iodophors - Iodine + detergent = BEST!  SODIUM POLYANETHOL SULFATE (SPS)
o Clorhexidine concentration: 0.025 % weight per volume; yellow (Other
o Hexaclorophene yellow contains a gel separator for chemistry)
o 10% hydrogen peroxide is used for cleansing  HEPARIN – for viral cultures, (not suitable for bacterial)
wounds. inhibits growth of gram pos bacteria and yeast.
II. Disinfectants – for non-living things NOTE: ACD, Citrate and EDTA anticoagulants are NOT used in
o Aldehydes – generally not used due to irritating microbiology (efficacy is not yet well researched)
fumes.
o Household bleach – ____________________ TRANSPORT MEDIA
o Tincture - With nutrients to maintain the growth of organism, buffer to
o Heavy metals – 1% silver nitrate maintain pH, small amount of gar to maintain moisture.
o Quaternary Ammonium Compounds -inactivated by
organic materials.  Stool pathogens: CARY BLAIR
o Phenolics – Amphyl
 Stuart’s – VTM
COLLECTION TRANSPORT & SPECIMEN PROCESSING  Amies - Modified stuart’s; Clinical swab
A. Specimen Collection and Transport  Transgrow – Neiserria spp.
TRANSPORT CONDITION TYPE OF SPECIMEN
 Collect before antibiotic therapy, ensure aseptic collection  Body fluids
and quantity must be sufficient, must be placed in sterile
Immediately at RT  Corneal scrapings
container.
Ambient RT in Microbiology:  Bone
 Ideally specimens should be transported to the lab within
22°C  Gastric Aspirate
2 hrs. of collection.
Immediately (within 15  Prostatic samples
minutes after collection)  Suprapubic aspirate
 For aerobic – swab, needle aspiration
 For anaerobic – needle aspiration, tissue  Tissue
 Urine, CSF, serous fluid, stool, sputum – sterile container  Gastric biopsy
Within 1 hour at RT
 Use sterile cotton tip applicator for throat, nose, eyes,  Stool
wound and abscess.  Abscess, lesions,
 Sterile bottle broth with SPS for blood culture wound.
 Blood
B. Anticoagulants/ preservative  BMA
 Preservatives can be used when transport of specimen is Within 2 hours at RT  Genital Tract
expected to be longer than 2 hours. specimens
 BORIC ACID – for urine (maintain quantity and qualities of  Respiratory Tract
colonies) specimens
 _________________________________________–  Urine
for stool (prevents stool from drying)

9
  Urine samples from  INDICATOR: ______________in the absence of air, they
Within 2 hours at 4°C
Foley catheter become COLORLESS
 Hair, nails, skin  Candle jar (seen in lots of microbiology laboratory)
scrapings (Fungal enclosed burning candle and tightly close it (use of oxygen
culture) and in the jar in increase carbon dioxide)
If with transport
Within 24 hours at RT
medium/preservatives:
 Stool
 Rectal swab
 Urine

SPECIMEN STORAGE
STORAGE CONDITION TYPE OF SPECIMEN
CSF (except for viral studies;
prolong viability by storing at ref.
37°C temperature or higher temperature
for longer storage)

Urine Stool
Viral specimen
Swabs
4°C
Foreign devices (ex.
Catheters)

-20°C Serum (for 1 week)

Tissues
-70°C Specimens for long term
storage

GASPAK JAR
 Supports growth of organisms (anaerobes, capnophiles,
microaerophiles) CLINICAL SPECIMEN
 Anaerobic jar uses gas generator increase hydrogen and GENERAL COLLECTION INFORMATION
carbon dioxide, contains pellets at top of container FOR ABSCESS, LESIONS, WOUNDS
(catalyst of oxygen reduction); Has indicator (with Container Swabs (aerobic; most common; poor
methylene blue) of presence of oxygen (turns blue) type of specimen because it is prone
to contamination and drying
10
 out; recommend needle URINE
aspirate/collect tissue) or FOR URINE: (commonly sent to the laboratory especially if
sterile tube (for aspirates) clinicians expect urinary infections)
Patient Preparation Wipe around the area with Container Clean, screw cap container
sterile saline or 70% alcohol (leak proof)
Special Instructions Swab along the leading edge of Patient Preparation Clean genitalia with soap and
the wound (find the main source of water (preferably first morning
infection) because it is more
NOTE: Swabs are considered poor specimen. Preferably, concentrated)
collect tissue or needle aspirates. (Especially for deep Special Instructions MIDSTREAM, clean catch
wounds)  May use as preservative: BORIC ACID
 Major cause of UTI: E. coli; in young females: S. sapro
WOUNDS/ABSCESSES  Suprapubic urine for anaerobic culture
1. Superficial skin infections: Staphylococcus aureus and  Colony count should be performed on all urine sample,
Streptococcus pyogenes when performing colony count use CALIBRATED LOOP
2. Folliculitis (hair follicle infection): S. aureus and (1µl or 10µl)
Pseudomonas aeruginosa  Formula to compute for colony count per mL of urine.
3. Boils, bedsores, etc.: S. aureus  # of colonies counted x 1000 (for 1µl) colony count/mL
4. Impetigo: S. pyogenes and S. aureus  # of colonies counted x 100 (for 10µl) colony count/mL
5. Erysipelis: S. pyogenes and less commonly
 CONSIDERED SIGNIFICANT: >100,000 or >10 5
Erysipelothrix rhusiopathiae
CFU/mL.
6. Deep and surgical wounds and abscesses: Anaerobes
from normal body sites

11
12
 o CONSIDERED SIGNIFICANT: >100,000 or >10 5 CFU/mL

 Bacteriuria is bacteria in the urine, but it may not indicate THROAT SWAB
a UTI.  Medium of choice: Sheep’s Blood Agar
 Calibrated loops are used to determine colony counts on  Most abundant throat flora: Viridans group Streptococci
media.  S. pyogenes (group A Streptococcus) - Most
 Urine specimens are generally plated onto SBA and MAC common throat pathogen
or EMB.  Group B streptococci.
 Common significant urine isolates include:  Group C streptococci.
o E. coli,  Group G streptococci and
o Klebsiella spp.,  Arcanobacterium spp
o Enterobacter spp.,
o Proteus spp.,
o Staphylococcus aureus,
o Staphylococcus saprophyticus,
o Enterococcus spp.,
o Pseudomonas aeruginosa
o Yeast.

13
 SPUTUM
FOR SPUTUM: (dealing with pneumonia, tuberculosis)
Container Clean, screw cap container
Patient Preparation Deep cough into a sterile
container
Special Instructions FIRST MORNING
SPECIMEN
 Non-sterile specimen
 May often be contaminated with normal flora so it is
important to evaluate the quality of spx.
 >25 PMN, <10 Squamous epithelial cell – suited for STOOL
culture FOR STOOL: (diarrhea, gastroenteritis: know the pathogen
 A gram stain is performed on all sputum sample; Collected responsible for the infection)
ideally in AM when it is most concentrated. Container Clean, screw cap container
Patient Preparation Excrete directly to the
TYPES OF COLLECTION: container (for bacterial
 EXPECTORATED (deep cough into a sterile container) culture: stool culture once a
 INDUCED SPUTUM COLLECTION (patient will inhale a day for 3 days; for babies:
solution which stimulates coughing) rectal swab as long as there is
 ETA – Sample to be collected if patient is intubated. visible fecal material around
the area)
GENERAL COLLECTION INFORMATION FOR SPUTUM Special Instructions PEA-SIZED specimen
 A direct Gram stain is performed to determine the quality  Detection of enteric pathogen
of the specimen.  Stool specimen not processed within 2 hrs of collection
 Acceptable specimens are cultured on SBA, MAC, and should be placed on transport media like Cary Blair.
chocolate agars.
 <10 squamous epithelial cells and >25 PMNs/low 1. Feces contain many species of anaerobic and facultative
power field anaerobic normal flora.
2. Bacteria causing gastroenteritis include:
 Streptococcus pneumoniae  Shigella spp.,
 Klebsiella pneumoniae  Salmonella,
 Staphylococcus aureus  Campylobacter jejuni,
 Pseudomonas aeruginosa  E. coli (e.g., O157:H7),
 Haemophilus influenzae  Yersinia enterocolitica,
 Legionella pneumophila  Clostridium difficile (must test for cytotoxin)
 Mycoplasma pneumoniae  Vibrio spp.
3. 4 Quadrants in Plated media

14
  Brucellosis – 3-4 weeks
 Leptospirosis – 8 weeks

BLOOD
FOR BLOOD:
Container Blood culture media set
(aerobic or anaerobic) Signs of growth: Bubbles, Hemolysis, cloudiness, pellicle
(different colors depending on
the tests) CEREBROSPINAL FLUID
Patient Preparation Disinfect site with 70% alcohol  Mode of collection: Lumbar puncture in between L3 and
and Povidone iodine L4, OR L4 and L5
Special Instructions Draw blood at the time of  Up to 20 mL of CSF can be collected.
febrile episodes (having  Common pathogens: Haemophilus influenzae, Neiserria
fever) meningitidis
Collect specimen at 2
different sites (increase
chances of getting pathogens
responsible for infection)
 Avoid normal skin flora while collecting blood culture.
 Blood culture bottles often contain 0.025% sodium
polyanethol sulfonate (SPS), an anticoagulant that also
inhibits complement and inactivates neutrophils.
 Dilution factor of blood to medium 1:10
 TAT: 5-7 days
 Adult: 10- ml (5 mL each site); Pedia 1- 5 mL
 The volume of blood collected probably has the greatest
effect on isolation of bacteria.
15
  Tube 1 = least affected by blood or bacteria introduced
ORDER OF TUBE COLLECTION AND STORAGE by spinal tap
1. Chemistry, Serology - Frozen (C.S.F)  Tube 2 = usually designated to microbiology
2. Microbiology - Room Temp (M.R.T)  Tube 3 = least likely contain cells introduced by spinal tap
3. Hematology - Refrigerated (H.R)  Tube 4 = better exclusion of skin contamination or for
additional test (Micro/Serology)

What if 1 tube only was collected?

Microbiology → Hematology → Chem/Sero

Receiving Day 0 – Culture Day 1 – Culture with

of Growth
Specimen

16
 Day 2 – Identification and
Day 3 – Reporting
Susceptibility

Day 1-5 – Culture with Growth

BD Phoenix M50
BD Bactec
Automated Blood Culture Automated ID AST

BACTERIAL IDENTIFICATION
1. Morphology/ appearance
 Consider the ff; size, shape, arrangement, motility and
staining characteristics
 MOTILE – Those with Flagella
 NON – MOTILE – No flagella
Blood
17
 1. Weigh
2. Dissolve
3. Dispense
4. Sterilize

*Water to make agar: Deionized/Distilled water

PREPARATION OF BLOOD AGAR PLATE

STUDY OF CULTURAL / COLONIAL CHARACTERISTICS TUBED MEDIA

(PHENOTYPICAL) CHARACTERIZATION 1. In 1 liter of distilled water, dissolve 28 g of nutritious agar
powder.
Culture media – contains the nutritional requirements needed for 2. To properly dissolve all components, heat this mixture while
bacterial growth stirring.
Culture - growth of microorganism in culture medium. 3. For 15 minutes, autoclave the dissolved mixture at 121
Types of culture: degrees Celsius.
 Pure culture – Contains 1 spp. of organism 4. Allow the nutrient agar to cool but not solidify after autoclaving.
 Mixed culture – Contains >1 spp. of organism 5. Add 5 percent (vol/vol) sterile defibrinated blood that has been
 Stock Culture – Research purpose – ATCC warmed to room temperature to the chilled agar and mix gently
but thoroughly.
6. Air bubbles should be avoided.
7. While still liquid, dispense into sterile plates.

STEPS TO PREPARE MEDIA

PLATED MEDIA
1. Weigh
2. Dissolve I. According to Physical state/Consistency
3. Sterilize
4. Dispense  LIQUID – No solidifying agent/ Agar, 0% agar,
Example:BHI,Nutrient broth, Alkaline peptone
18
  SEMI –SOLID – 0.5 – 1% agar  Enriched media
Example: SIM  Enrichment media
 SOLID – 2-3% agar  Selective media
o Liquefiable – When heated again, it liquifies.  Indicator media
Example: MAC, SSA, EMB  Differential media
o Non-liquefiable – when heated, it no longer  Sugar media
liquefies.  Transport media
Example: Rice agar  Media for biochemical reactions
o Biphasic – Both solid and liquid media
Example: Castaneda (for Brucella)

General purpose media/ General isolation media – contains

only the necessary nutrition to support bacterial growth, for
Agar – usually derived from red algae, solidifies at 40-50 degC,
routine cultivation of bacteria.
metls at 80 -90degC. Cooling temperature for distribution of
media to plate is 55 – 60 degC.
Example:
Amount: 25mL / plate
Nutrient agar and broth
II. Based on the constituents/ ingredients
 Simple medium
 Complex medium
 Synthetic or defined medium
 Special media

III. According to use:

 General

19
Simple media / basal media
 Eg: NB, NA
 NB consists of peptone, meat extract, NaCl,
 NB + 2% agar = Nutrient agar

Complex media
 Media other than basal media.
 They have added ingredients.
 Provide special nutrients

Synthetic or defined media

 Media prepared from pure chemical substances and its
exact composition is known Example:
• Eg: peptone water – 1% peptone + 0.5% NaCl in water Selenite F Broth – for the isolation of Salmonella, Shigella
Alkaline Peptone Water – for Vibrio Cholera

Enriched media
Substances like blood, serum, egg are added to the basal Selective media
medium.  The inhibitory substance is added to a solid media.
Used to grow bacteria that are exacting in their nutritional needs. Example:
Example: Sheep’s Blood agar, Chocolate agar  Mac Conkey’s medium for gram negative bacteria
 _________ – for [Link]
 _________ – [Link]
 Wilson and Blair medium – [Link]
 Potassium tellurite medium – Diphtheria bacilli

Transport media
 Media used for transporting the samples.
 Delicate organisms may not survive
the time taken for transporting the
Enrichment media specimen without a transport media.
Liquid media used to isolate pathogens from a mixed culture. o Stuart’s medium – non nutrient
Media is incorporated with inhibitory substances to suppress the soft agar gel containing a
unwanted organism. reducing agent
o Buffered glycerol saline –
enteric bacilli
20
  A single stain is used.
Multitest Systems  It is directed towards coloring the forms and shapes
• API present.
• BD BBL Enterotube II o Malachite Green, Methylene Blue, Crystal Violet,
• BBL Crystal System Carbol Fuchsin, Safranin
• Remel’s RapID o When reporting identifies the color reaction,
morphology, arrangement
Automated Identification Systems in Microbiology o Simile (single)
• Colorimetry – measure color change o There are no cluster bacilli
• Nephelometry – light scattering o Clusters are applicable to cocci
• Fluorometry – fluorescence o Using simple stain is not applicable for grouping

Automated systems for

Identification
• MicroScan System
• Sensitire System
• BD Phoenix
• VITEK System

CLASSES OF IONIZABLE DYES

1. BASIC DYES

 Are commonly dyes used in bacteriology

 Are cationic or have positively charged groups
(pentavalent nitrogen) that bind to negatively charged
molecules like nucleic acids and proteins (bacterial cell 2. DIFFERENTIAL STAINING
wall)
o Methylene Blue, Basic Fuchsin, Crystal Violet,  It divides bacteria into separate groups.
Safranin and Malachite Green  It is directed towards coloring components of those
2. ACID DYES elements present.
 Are anionic or possess negatively charged groups  Inoculums size for staining: 10^5 CFU/Ml
(carboxyls and phenolic) that bind to positively charged  Gram staining and Acid-Fast Bacilli (AFB)
cell structures. staining (colors the cell wall)
o Eosin, Rose Bengal and Acid Fuchsin STEPS IN DIFFERENTIAL STAINING: (uses more than one
stain)
STAINING TECHNIQUES a. Application of primary stain
1. SIMPLE STAINING b. Application of mordant
21
c. Application of decolorizing agent
d. Application of secondary stain /counterstain

GRAM STAINING

3. DIAGNOSTIC ANTIBODY OR DNA PROBE MEDIATED

STAINING

It is directed specifically at identification of

an organism.
It is used for the specific identification of
selected pathogens, such as:

Chlamydia trachomatis, Bordetella

pertussis, Legionella pneumophilia,
HSV, varizella-zoster virus, CMV,
adenoviruses and respiratory viruses.

4. NEGATIVE STAINING  Gram (+) Have thick Peptidoglycan and teichoic acid
contributing to the adherence of Crystal Violet; resist
 To demonstrate the presence of diffuse capsule decolorization
surrounding some bacteria  Gram (-) Have a thinner peptidoglycan and outer
 To study bacterial gas vacuoles and viral morphology lipopolysaccharide; will stain purple, dehydrate
 To study cells sensitive to heat fixing decolorized retain safranin
 Result: the bacteria appear as light-colored bodies against  Too much/long staining could result to false gram Negative
a dark background – the cell surface repels the acidic stain  Critical part in Gram staining is decolorization
since the bacterial cells are negatively charged
o India ink or Nigrosin dye (acidic stain)

22
 5. Failure to leave the safranin in place for sufficient time will
result in failure to stain the Gram-negative bacteria and
background.

ACID FAST STAINING

 Acid-fast stain is a differential stain used to identify acid-
fast organisms such as members of the genus
Mycobacterium. Acid-fast organisms are characterized by
wax-like, nearly impermeable cell walls; they contain
mycolic acid and large amounts of fatty acids, waxes, and
complex lipids.
 MYCOLIC ACID aka: Hydroxymethoxy acid
o The substance unique in the cell wall of AFO

ACID FAST STAIN

 It is used to stain bacteria that have high lipid content in
KEEP IT POSITIVE OR NEGATIVE? their cell walls.
Principle:
PRECAUTIONS DURING GRAM STAINING:  The primary stain binds to mycolic acid in the cell walls of
1. If the CV is rinsed too vigorously before it is complexed with the mycobacteria and is retained after decolorization with
the iodine, it will wash away and leave poor or no staining of Gram acid alcohol.
negative bacteria.  Acid-fast bacilli (AFB) retain the primary stain and are
2. If the decolorization is too vigorous or prolonged, the gram- colored red (carbol fuschin) and non-AFB blue or green
positive complex will be removed and the normally Gram-positive (methylene blue or malachite green counterstains) color.
bacteria will not stain. (False gram negative) (resistant to decolorization)
3. If the decolorization is insufficient, organism may appear falsely
Gram-positive.
4. If the safranin is left on the slide for a prolonged period
(minutes), the Gram-positive complex will be leached from the
23
  Non-Acid fast don’t contain mycolic acid and become  WAYS TO FACILITATE ACID FAST STAINING:
decolorized with alcohol  Distinguish AF from NAF organisms
 AFS -
1. STEAMING PROCESS - to temporarily remove mycolic
acid

MODIFIED ACID FAST STAINING METHOD:

 Is useful for the identification of intestinal coccidian
oocysts (Cryptosporidium and Cyclospora); modified
ACID FAST STAINING METHODS Kinyoun
1. Ziehl-Neelsen/Hot Staining Method – uses steam (physical  Specimen: stool (freshly collected or preserved in formalin)
mordant)  Reagents: same with the conventional acid-fast reagents
2. Kinyoun’s/Cold Staining Method – same reagent but omits except for the concentration of the acid alcohol (1%
heat (carbol fuchsin has higher concentration of phenol which H2SO4) (milder)
serves as a chemical mordant); sometimes have a wetting agent  Results: oocysts appear as magenta-stained organism
tergitol to fasten up reaction against a blue background
3. Pappenheim’s Method – differentiates M. smegmatis (blue)
from M. tuberculosis (red)
4. Baumgarten’s Method – differentiates M. leprae from M.
tuberculosis
5. Auramine-Rhodamine Method – selective for the cell wall of
AFB

Rule after AFB Staining:

24
ALL BACTERIA ARE NON-ACID FAST EXCEPT: Mycobacteria, SUSCEPTIBILITY TESTING
Nocardia, Rhodococcus, Tsukamurella, Gordonia and
Legionella micdadei. Susceptibility Testing
 To detect ability of antimicrobial agent to inhibit. Bacterial
(+) MODIFIED acid-fast staining growth in vitro
 To determine susceptibility/ resistance of organism against
PARASITES: antimicrobial agents.
______________________________
METHODS:
SPECIAL STAINS 1. Dilution method – standard inoculum for broth dilution 5 x 10 5
1. Dyar stain - Cell Wall CFU/mL; for agar dilution 1 x 10 4 CFU/mL
2. Hiss, Gin, Anthony and Welch - Capsule TOTAL BROTH VOLUME: Microdilution – 0.5 – 0.1 mL ;
3. LAMB, Neisser, Albert, Ljubinsky – Metachromatic granules Macrodilution 1 mL or >
4. Dorner, Wirtz and Conklin, Schaeffer-Fulton - Endospore
(common with Ziehl-Neelsen but different reagent) 1. 1. BROTH DILUTION/ TUBE DILUTION
5. Gray, Leifson, Fisher and Conn - Flagella
6. India Ink/Borris Method and Nigrossin - Capsule (yeast cells)  Challenging the organism with antimicrobial agents in
7. Feulgen Stains - DNA liquid environment (broth medium)
8. Fontana Tribondeau and Levaditi Silver Impregnation –  Concentration range for each antibiotic – doubling
dilutions
 Incubate 37 degC
 MIC (minimal inhibitory concentrations) – Lowest
concentration of antibiotic that inhibited bacterial growth
 MBC/MLC - Lowest concentration of antibiotic that killed
the organism

25
 ADVANTAGE – provide information to both:

QUALITATIVE RESULTS
QUANTITATIVE RESULTS

MICRODILUTION
 Most laboratories use commercially supplied microdilution
panels in which broth is already supplement appropriate
antimicrobial concentration, thus antimicrobial preparation
and dilution are not commonly carried out
 It is essential to prepare standard inoculum from a fresh
overnight pure culture of test organism
 Inoculated trace are incubated under optimal AGAR DILUTION
environmental conditions usually 35 – 37o C  Antimicrobial concentrations and organisms on an agar
based medium
BREAKPOINTS  Allows examination of one or more bacterial isolations per
 Specific concentrations that separate/define the different plate
categories  Each doubling dilution of an antimicrobial agent is
incorporated into a single agar plate
BREAKPOINT PANELS
 Panels that only contain antimicrobial concentrations
26
  Testing of a series of 6 dilutions of one drug requires six
plates plus one positive growth control plate without DISK DIFFUSION (KIRBY – BAUER)
antibiotic  Challenging bacterial isolates with antibiotic disks placed
on the surface of an agar plate that has been seeded with
• STANDARD INOCULUM SIZE: ____________________ a lawn of bacteria.
 Advantage: provides a means for determining MIC for
 Neisseria gonorrhoea, which does not grow  MEDIA: MHA
 sufficiently in broth  DEPTH OF AGAR: ________
 Reference method for testing anaerobes and  pH of agar: ___________
[Link]  Filter paper disk size: 6 mm
 Reference method for anaerobes: Brucella agar with laked MHA with 5% blood – Strep and N. meningitidis
blood and vitamin K (Wadsworth method) MHA with 2% Nacl – MRSA and staph

E TEST
 Is a dilution test based on the diffusion of a continuous INOCULUM PREPARATION
concentration gradient of an antimicrobial agent from a
plastic strip into an agar medium. USE OF PURE CULTURE 4 – 5 colonies inoculate into
 (+) result: ____________ broth medium incubate for 3 –
 An alternative susceptibility test for fastidious bacteria (S. 5 hours at 35 – 37oC
pneumonia and H. influenza). 4 – 5 colonies, 16 – 24 hours
suspend in broth or 0.9%
saline solution (bacterial
suspension/ organism
suspension)
USE OF STANDARDIZED – Turbidity Standard:
size of Inoculum  ________

27
  (Barium Sulfate
Suspension)
Composition:
 99.5 ml of 1% Sulfuric
acid
 0.5 ml of 1.175%
Barium chloride
Standard Size of Inoculum:
1.5 x 108 CFU/ml
READING AND INTERPRETATION OF RESULTS
1. Examine for the presence of confluent lawn of growth
a. If POOR or NONCONFLUENT – repeat testing
2. Examine for purity
a. If MIXED CULTURES – repeat testing
3. A dark background and reflected light are used to examine the
plate
4. Measure the zone of inhibition in diameters for each
antimicrobial agent using a ruler or caliper
5. Interpret as “SUSCEPTIBLE”, “INTERMEDIATE” or
“RESISTANT”

CAUSES OF FALSE RESISTANCE

INOCULATION AND INCUBATION • Use of heavy inoculum or undiluted
1. Surface of the plate is swabbed in three directions (submerged specimen
in bacterial suspension standardized to match turbidity of 0.5% • Late examination of plates
Mcfarland Standards) • Use of disk with inadequate drug
2. Antibiotic disks are applied within 15 minutes concentration
3. Inverted plates – incubation (prevent accumulation of moisture) • Failure to immediately refrigerate the
4. Incubate at _______ disk after use
5. Increased CO2 for __________ • Use of wrong bacterial isolate
6. Incubation time may be increased beyond 16 hours – for
methicillin resistance staphylococci and vancomycin resistance FACTORS AFFECTING THE ZONE OF INHIBITION
enterococci
Amount of inoculum or  Only pure cultures can be
test organism tested
 Too light – false
susceptibility
 Too heavy – false
28
 resistance Concentration of divalent  Affect testing of
Thickness of the agar  Ideally _________ cations Aminoglycosides and
 Too thick – smaller zone Tetracycline against
size Pseudomans
 Too thin – __________ aeruginosa.

Growth rate of the test  35 degrees C for 16-24 WHAT IS D TEST?

organism hours (recommended for  CLSI recommendation to detect inducible clindamycin
most) resistance among strains of SAU, Strep and other B
 >35 degrees C – false hemolytic.
detection (MRSA)  Uses 2 antibotics: 15 ug Erythromycin, 2 ug Clindamycin
 <35 degrees C – larger  Blunting or flattening of Clindamycin
zone of inhibition
 Increased CO2 for
fastidious bacteria
o Neisseria
meningitis
o Streptococcus
pneumoniae
 7.2 – 7.4 pH
 Incubation of plates in
CO2 – decrease pH
 High pH – decrease
activity of some
antimicrobial
o Tetracycline
 Low pH – diminished
The Ph of the medium activity of some
antimicrobial
o Aminoglycosides
o Erythromycin
Number of disk per plate  150 mm plate can have
maximum of 12 disk
 More than 12 disk –
overlapping zones
leading to errogenous
results
29
 ANTIMICROBIAL AGENT GRAM STAINS 1. Neiserria
2. Veilonella
3. Moraxella
4. Branhamella

ALL BACILLI ARE GRAM NEGATIVE EXCEPT

“CALL BC MEN”

1. Clostridium
2. Actinomyces
3. Lactobacillus
4. Listeria
5. Bacillus
6. Corynebacterium
7. Mycobacterium
8. Erysiphelothrix
9. Nocardia

ANTIBIOTIC CLASSES (MNEMONIC) GRAM POSITIVE COCCI

“Antibiotics Can Terminate Protein Synthesis For Microbial FAMILY MICROCOCCACEAE

Cells Like Germs”
• Staphylococci
1. Aminoglycosides • Micrococci
2. Cephalosporins • Stomatococcus (animals)
3. Tetracyclines • Planococcus (animals)
4. Penicillins • Streptococci
5. Sulfonamides 1. Gram staining:
6. Fluoroquinolones STAPHYLOCOCCUS - G(+) cocci in clusters
7. Macrolides MICROCOCCI - G(+) cocci in tetrads (by 4) / sarcina (by 8)
8. Carbapanems
9. Lincosamides 2. Growth on BAP 5% Sheep's blood
10. Glycopeptides (Substitute: horse's blood, rabbit's blood)

ALL COCCI ARE GRAM POSITIVE EXCEPT 3. Growth on Loeffler’s Serum Slant
Purpose: enrichment of the pigment production of
“Never Mind the Boys” Staphylococcus

30
 S. aureus - golden yellow pigment
S. citreus - lemon yellow pigment S. aureus - yellow halo formation
S. albus - porcelain white pigment S. epidermidis & saprophyticus - pink colonies

4. Growth on MSA (Mannitol Salt Agar)

Inhibitor: 7.5% NaCl
Carbohydrate: Mannitol pH
indicator: phenol red
*acidic / positive result - yellow
*alkaline / negative result - red
S. aureus – yellow halo formation
S. epidermidis – pink colonies
S. saprophyticus – pink colonies

5. Catalase Test
- Differentiates Staphylococcus and
Streptococcus
Reagent: 3% H2O2
(+) result: bubbling formation / effervescence / gas bubbles 3% TESTS TO DIFFERENTIATE STAPHYLOCOCCUS AND
2H2O2 --- Catalase ---> 2H2O + O2 MICROCOCCUS
(+) Staphylococcus and Micrococcus (-)
Streptococcus PROPERTIES STAPHYLOCOCCUS MICROCOCCUS

1. Aerobic
growth
2. Anaerobic
growth
3. Lysostaphin
4. Bacitracin /
Taxo (0.40
U)
5. Modified
Oxidase
6. Glucose Fermentation > C > Oxidation > O2
GROWTH ON MSA (MANNITOL SALT AGAR) Utilization Acidic > Alkalinic
Inhibitor: 7.5% NaCl AKA: O/F test
Carbohydrate: Mannitol pH
indicator: Phenol red 7. Furazolidone S R
- acidic/positive result: YELLOW
- alkaline/negative result: RED
31
*Staphylococcus – facultative anaerobe
*Micrococcus - obligate aerobe

Additional Notes:
*Stomatococcus – Modified Oxidase (-), Lysostaphin (R)
and Furazolidone (R)
*Staphylococcus is more resistant to lysozyme (destroys
peptidoglycan)
Tests to differentiate Staphylococcus and Micrococcus
6. Modified Oxidase Test  Enzyme that can possibly dissolve clot: fibrinolysin /
Reagent: Staphylokinase
1% tetramethylparaphenylenediaminedihydrochloride in
dimethylsulfoxide Staphylococcus aureus
(+) result: blue / purple (Micrococcus luteus)  Gram positive cocci arranged in clusters
(-) result: no color change  Protein A – cell wall, anti-phagocytic, virulence
Indicator: bromthymol blue  β hemolytic colonies or non hemolytic on BAP
 CNA with cream to golden yellow pigmentation
*Alkalinic - blue  Normal flora of skin
*Acidic – Yellow  Boils, carbuncles
 Associated w/ foreign things (e.g. tampon)
7. ______________  #1 wound infection; #1 osteomyelitis (septic arthritis)
Fibrinogen ------> Fibrin clot ◻ Most definitive test / determinant
 Pigment: cytochrome / staphyloxanthin (golden yellow
to S. aureus (+)
 pigment)
A. Slide Coagulase (Screening test)
Detects: cell bounded coagulase / clumping factor  Inhibited by bile
Reagent: rabbit plasma (must be obtained using EDTA top  MRSA (Methicillin-Resistant Staphylococcus aureus) –
tube) grows
(+) result: clot/clumping  in 35◦C with 2% NaCl; Treatment: Vancomycin
Bound coagulase (clumping factor): POSITIVE
B. Tube Coagulase (Confirmatory test) Free coagulase: POSITIVE
Detects: _______________ MSA: Growth and Fermentation
Reagent: rabbit plasma (must be obtained DNAse: _______________
using EDTA top tube) Thermostable nuclease: POSITIVE
(+) result: clot/clumping
Important rule: check every 30

32
 • Vibrio cholerae
• Stenotrophomonas maltophilia
• Staphylococcus aureus

Medium: DNA medium ∙

Two methods:
Toluidine blue – (+) = pink zone
Methyl green – (+) = clear zone
c. HCl precipitation – no precipitation after 1N HCl when
Differentiation among Coagulase (+) Staphylococcus DNase(+)
(+) = clear zone; (-) = no clearing
SPECIES COAGULAS E VP PYRASE
(ACETOIN)

1. Staphylococcus (+) (+) (-)

aureus spp.
Aureus
2. Staphylococcus (+) (-) (+)
intermedius 9. Novobiocin
3. Staphylococcus (+) (-) (-) Test (5μg)
hyicus Differentiates: Coagulase (-) Staphylococcus;
4. Staphylococcus (+) (+) (+) Identification of Staphylococcus saprophyticus
shleiferi
Staphylococcus saprophyticus
Other Staphylococcus aureus-like bacteria  Causes UTI in young women; Specimen for diagnosis:
*Staphylococcus lugdunensis – Slide coagulase (+); PYRase urine
(+)  Novobiocin RESISTANT (less than 16mm)
*Staphylococcus haemolyticus – β-hemolytic; Coagulase (-)
Staphylococcus epidermidis
8. DNase test  Gram positive cocci in clusters
Detects DNase (deoxyribonuclease) – degrades DNA Bacteria  White, creamy, raised growth on Blood agar
which are DNase test (+): “SMASH VSS”  Nonhemolytic on Blood agar
 CONS, Normal flora of the skin
• Serratia marcescens  DNAse negative
• Moraxella catarrhalis  Novobiocin SUSCEPTIBLE
• Aeromonas  Causes endocarditis (associated with Prosthetic Heart
• Streptococcus pyogenes Valve Surgery infection)
• Helicobacter pylori
33
 SUSCEPTIBLE

S. epidermis

RESISTANT

S. saphrophyticus

POSITIVE PATHOGEN DETERMINANTS OF S. aureus

SUMMARY:
1. Protein A - ___________
CATALASE TEST 2. Coagulase - converts fibrinogen to fibrin clot (can cause DIC)
Disseminated Intravascular Coagulation
3. Staphylokinase/fibrinolysin – dissolves fibrin clot
Reagent: 3% H202
4. Lipase - degrades lipids (cellulitis,boils)
5. Hyaluronidase - ____________________
+ : Bubbling NEGATIVE 6. __________- degrades DNA
7. Exfoliatin (epidermolysin)- destroys stratum
granulosum/skin; SSS - Scalded skin syndrome
8. Leukocidin - destroys PMNs and monocytes; Panton
Valentine factor
9. Hemolysin - destroys RBC; anemia
10. Enterotoxin - causes FOOD POISONING and SEPTIC
SHOCK SYNDROME; makes S. aureus the #1 cause of food
POSITIVE poisoning in the PH & US
11. TSST-1 (Toxic Shock Syndrome Toxin-1) - TAMPONS
COAGULASE 12. Beta lactamase
13. Gelatinase
NEGATIVE
Reagent: RABBIT
PLASMA

+ : Clumping

34
FAMILY STREPTOCOCCACEAE
 Genus Streptococcus; Gram (+) cocci in pairs/chains BLOOD AGAR:
 S. pneumoniae – G(+) diplococci; “lancet-shape”; "dome-
shape colonies” Shows three types of hemolysis:
 NON-MOTILE Alpha Hemolysis
Beta Hemolysis
 NON-SPOREFORMERS
Gamma Hemolysis
 Facultative anaerobe
 Catalase (-), white “pinpoint” colonies on BAP
 Capnophilic (5-10% CO2);
 Medium of choice: Sheep’s Blood Agar Plate
 Selective medium: PEA (Phenyl Ethyl Alcohol Agar)
 Susceptible to Vancomycin

Classification:

 Lancefield grouping – based on the antigens
(polysaccharides) present on the surface I. Lancefield grouping
 Smith and Brown’s grouping – based on hemolytic A. Streptococcus pyogenes
reactions B. Streptococcus agalactiae
 Bergey’s grouping – based on temperature C. Streptococcus dysagalactiae “D.E.E.E.Z”
 Streptococcus equisimilis
 Streptococcus equisimilus
 Streptococcus equinus
 Streptococcus zooepidimicus
D. Enterococcus “F.F.A.D”
 Enterococcus faecalis
 Enterococcus faecium
35
  Enterococcus avium  Hyaluronidase
 Enterococcus ducans  Sensitive to Bacitracin (Bacitracin / Taxo A positive
E. Non-Enterococcus control)
 Streptococcus bovis – Colon Cancer  Resistant to SXT – PYRase positive
 Streptococcus equinus  BETA HEMOLYTIC

II. Brown’s grouping Diseases:

Alpha Streptococcus pneumonia, Viridans, some of  flesh-eating bacteria
Hemolysis group D  Impetigo
Beta A, B, C, some of D  Pharyngitis
Hemolysis  RHD (Rheumatic Heart Disease) / RF (Rheumatic
Gamma Most of D Fever)
Hemolysis  AGN (Acute Glumerulonephritis)
 Scarlet fever
III. Bergey’s grouping  Erysipelas
a. Pyogenic – pus-producing Streptococcus spp.  Wound, burn
b. Lactic – Streptococcus spp. used in making dairy products  Toxic Shock Syndrome, pyoderma, necrotizing fasciitis
c. Enterococcus – normal flora of the gut
d. Viridans – normal flora of upper respiratory tract

Smith and Brown’s Grouping

Group B Streptococcus (S. agalactiae)

 β-hemolytic
Group A Streptococcus (S. pyogenes)  normal flora of vagina and upper respiratory tract
 M-protein – cell wall, anti-phogocytic, virulence factor  neonatal sepsis
 Streptolysin O – O2 labile, Antigenic, anerobic, lytic  #1 neonatal meningitis and (pneumonia, bacteremia)
 Streptolysin S – O2 stable, Non-antigenic  Resistant to Taxo A, Taxo P and SXT, Bacitracin
 Erythrogenic toxin – Scarlet feverStreptokinase  CAMP POSITIVE
36
 (+): arrow-head zone of β-hemolysis
Group C, F AND G– rarely cause disease (+) for: S. agalactiae, Listeria monocytogenes, Helicobacter pylori
 β-hemolytic
 Causes severe pharyngitis followed by bacteremia 4. Sodium-Hippurate Hydrolysis Test
 BACITRACIN RESISTANT (small percentage are  Indicator: Ninhydrin reagent (reacts to glycine)
susceptible)  Hippuric acid-------> benzoin + glycine
 SXT SUSCEPTIBLE
 Opportunistic pathogen that may also be associated with (+): purple / violet
pneumonia, cellulitis and abscess. (+) for: S. agalactiae

Group D – rarely cause disease 5. Bacitracin and SXT (Sulfamethoxazole and

 UTI and wound infection Trimetoprim)

1. Bile Esculin Hydrolysis Test

 Differentiates Group D from other Streptococcus,
presumptive test for Group D
 Medium: BEA (Bile Esculin Agar) – contains
 40% bile and esculin
 Indicator: FeCl3 (reacts to esculetin)
 40% bile + esculin-------> esculetin
RECALL QUESTION
(+) result: brown or black precipitate / blackening
(+) for: Group D Streptococcus The PYR hydrolysis test is a presumptive test for which
Streptococci?
6.5 % NaCl PYRase A. Group A and D (Enterococcus) Streptococci
E (+) (+) B. Group A and B Streptococci
NE (-) (-) C. Gardnerella vaginalis
D. NOTA
2. PYRase (Pyrolidonylarylamidase) test
 Substrate: PYR
 PYR ---PYRase---> β-naphthalidamide
 Color indicator: P-dimethylaminocinnamaldehyde (+): red

(+) for: Group D (Enterococcus), Group A

3. CAMP (Christine Atkins Munch Peterson)

 Principle: CAMP factor enhances β-hemolysis
37
 RECALL QUESTION

Positive control for Bile Esculin Agar

A. P. aeruginosa Streptococcus pneumoniae (Diplococcus pneumoniae)
B. Enterococcus faecalis  Normal flora of nasopharynx and oropharynx
C. S. pneumoniae  has capsule (virulence factor)
D. S. pneumoniae  not included in the Lancefield grouping
 ______________
 causative agent of Lobar pneumonia / pneumococcal
pneumonia (#1 community acquired pneumonia in the
Philippines)
 #1 adult bacterial meningitis
 otitis media (most common cause)

38
Viridans Schultz-Charlton Test – a skin test for scarlet fever that
 Normal flora of upper respiratory tract, gastrointestinal uses antitoxin to the erythrogenic toxin of S. pyogenes
tract and genitourinary tract subcutaneously; a positive reaction is blanching of the rash in
 N ot included in the Lancefield grouping the area around the injection site.
 -α hemolytic colonies
 SBE (Subacute Bacterial Endocarditis) Nutrionally Variant Streptococcus
>>>MUSCISM >requires cysteine for survival
also: Thiol, Vit. B6 (pyridoxine)
 Streptococcus mutans – dental plaques / caries e.g. Streptococcus defectivus Streptococcus adjacens
 Streptococcus uberis Abiotrophia spp.
 Streptococcus salivarius *shows satelliting on agar
*Staph Streak Test (+)
 Streptococcus constellatus
 Streptococcus intermedius
Pediococcus / Leuconostoc (Streptococcus-like
 Streptococcus sanguis organisms)
 Streptococcus mitis (mitior)  Vancomycin resistant test – differentiate Pediococcus from
Streptococcus viridans
Tests To Differentiate Streptococcus pneumoniae and
Viridans
TEST STREPTOCOCCUS VIRIDANS
Mouse Inoculation Dead Alive
Inulin fermentation (+) (-)
Bile Solubility (+)
(+) = due to
Result: clearing of the (-)
autolysis
medium
Optochin/Taxo P
[Composition:
S R
ethylhydroxycuprein
HCl; 5 ug]
Neufeld-Quellung [(+)
result: Capsular (+) (-)
swelling]

 Francis Test – detects pneumococcal antibody; skin test

for S. pneumoniae infection
 Dicks Test – detects erythrogenic antigen; skin test for S.
pyogenes infection; (+) result: redness
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 (+): Neisseria, Moraxella, Aeromonas, Pseudomonas

Techniques in performing the oxidase test:

a. Put a drop of reagent on the colony
b. Rub on a filter strip paper and add a drop of reagent
c. Rub the colony on a piece of filter paper containing the
reagent

Superoxol Catalase Test

Reagent: ____________
(+) vigorous bubbling formation
I. Neisseria gonorrhoeae
GRAM NEGATIVE COCCI  Kidney (coffee) bean shaped in PMN
GRAM NEGATIVE COCCI  Oxidase positive and ferment glucose
NEISSERIA  Virulence – “pili”
 G(-) diplococci EXCEPT Neisseria elongata
 Has pili Diseases:
 __________  Gonorrhea
 Fastidious organism  __________________
 Capnophilic (requires 5-10% CO2) Non-Motile  Cervitis, Salphyngitis (Female)
 Catalase(+) except N. elongata  Anorectal gonorrhea (Homosexuals)
 Oxidase (+) Superoxol (+)  Opthalmia neonatorum - blindness
 JEMBEC system – _______________  Pharyngitis (acquired through oral sex)
 Fitz-Hugh Curtis
 PPNG (Penicillinase Producing Neisseria gonorrhea)

Oxidase Test / Taxo N

 Presumptive (screening) test for gram negative cocci
Reagent: 1% tetramethylparaphenylenediaminedihydrochloride II. Neisseria meningitidis
(+) result: purple  Carrier: nasopharynx
 Virulence: capsule and endotoxin
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  Serotypes: A, B, C, Y, W135 (Capsular Antigens) ANTIBIOTICS
 Neufeld Quellung Test  Vancomycin - inhibits G(+) Colistin - inhibits G(-) Nystatin
- inhibits fungi
Diseases:  Modified TMA – VCNT (Vancomycin, Colistin, Nystatin,
 Meningitis Trimethoprim lactate)
 Meningococcemia – can lead to DIC (Disseminated  NYC Agar – VCAT (Vancomycin, Colistin, Amphotericin B,
Intravascular Coagulation) Trimethoprim
 Waterhouse-Freiderichsen Syndrome – disease of the  lactate) ∙ Martin-Lewis Agar – VCAT (Vancomycin, Colistin,
adrenal glands Anisomycin, Trimethoprim lactate)

Cultivation:
 CAP (+)
 BAP (-) Cystine Trypticase Agar
 GC (Gram Negative Cocci) Agar – AST media  Different carbohydrates in CTA tubes are oxdized into
 Thayer-Martin Agar (selective) organic acids that change phenol red into a yellow color
 Modified Thayer-Martin Agar  Positive: yellow color in the upper layer of the medium
 New York City Agar (Mycoplasma hominis and
Ureaplasmaurealyticum can also grow)
 Martin-Lewis Agar

Composition:
 Base medium: CAP
o Thayer-Martin Agar - VCN

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 utilization tube reactions observed in the lower photograph, what
is the presumptive identification of this isolate.

A. Neisseria gonorrhoeae
B. Neisseria meningitidis
C. Aeromonas species
D. Kingella kingae

CARBOHYDRATE FERMENTATION/UTILIZATION TEST

RECALL QUESTION III. Other Neisseria – rarely cause infection

 Wound infection
Small intracellular gram-negative cocci were observed in a  Pneumonia
centrifuged spinal fluid sediment as presented in the upper  UTI
photomicrograph. A clinical diagnosis of meningitis was
suspected. Yellow-gray colonies after 24-hour incubation had IV. Moraxella catarrhalis (Old name: Neisseria catarrhalis or
been observed on blood agar. Based on the carbohydrate Branhamella catarrhalis) – rarely cause infection
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 Gram (-) diplococci (Morphologically and biochemically resembles Neisseria
 Oxidase positive, reduce nitrate to nitrite, DNase positive
(best test to differentiate from other Moraxella)
 Tributyrin Hydrolysis (Butyrate Esterase Disc Test) = (+)
 Assacharolytic
 Pneumonia and other upper respiratory tract infections
 Bacteremia
 3rd cause of otitis media (middle ear infection)

REFERENCES:

1. Mahon, C. R., Lehman, D.C., & Manuselis, G. (2015).

Textbook of Diagnostic Microbiology 5th Edition. Maryland
Heights, MO. Saunders Elsevier.
2. Forbes, B.A., Sahm, D.F., & Weissfeld, A.S. (2007). Bailey
& Scott’s Diagnostic Microbiology 12th Edition. St. Louis,
MO. Mosby Elsevier.
3. Delost, M.D., (2015). Introduction to Diagnostic
Microbiology for the Laboratory Sciences. Burlington, MA.
Jones & Bartlett Learning.
4. Special mention to Doc Liwanag’s NOTES <3
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