MICROBIOLOGY

PRACTICAL

ASHRAUL KARIM
DIPTENDU MAJUMDER
 BACTERIA

MICROSCOPIC
 • Gram-positive bacteria: Stain purple/blue due to thick peptidoglycan
wall retaining crystal violet.

• Gram-negative bacteria: Stain pink/red due to thin peptidoglycan

and outer membrane; they lose the crystal violet and take up the
safranin counterstain.

GRAM STAIN

Procedure

Fixation: The smear made on a slide from bacterial culture or specimen is

air-dried and then heat-fixed.

Step 1 (Primary stain): The smear is stained with pararosaniline dyes such
as crystal violet (or gentian violet or methyl violet) for one minute. Then
the slide is rinsed with water. Crystal violet stains all the bacteria violet
in color (irrespective of whether they are gram-positive or gram-
negative)

Step 2 (Mordant): Gram’s iodine (dilute solution of iodine) is poured over

the slide for one minute. Then the slide is rinsed with water. Gram’s
iodine acts as a mordant, binds to the dye to form bigger dye-iodine
complexes in the cytoplasm.

Step 3 (Decolorization): Next step is pouring of few drops of decolorizer

to the smear, e.g. acetone (for 1–2 sec) or ethyl alcohol (20–30 sec) or
acetone alcohol (for 10 sec). Slide is immediately rinsed with water.
Decolorizer removes the primary stain from gram-negative bacteria
while the gram-positive bacteria retain the primary stain.
Step 4 (Counter stain): Secondary stains such as safranin or dilute carbol
fuchsin is added for 30 seconds. It imparts pink or red color to the
gram-negative bacteria. Alternatively, neutral red may also be used as
counter stain, especially for gonococci. The slide is rinsed in tap water,
dried, and then examined under oil immersion objective.

Interpretation of Gram Stain

Smear is examined under oil immersion objective.

Gram-positive bacteria resist decolorization and retain the color of

primary stain i.e. violet

Gram-negative bacteria are decolorized and, therefore, take counterstain

and appear pink.
Principle of Gram Staining

The Gram staining technique differentiates bacteria into Gram-positive

and Gram-negative based on their cell wall properties and how they
interact with dyes. While the exact mechanism is not fully understood,
two main theories explain the differential staining:

1. pH Theory
• The cytoplasm of Gram-positive bacteria is more acidic, which
enhances retention of basic dyes like crystal violet.
• Iodine acts as a mordant, forming a crystal violet–iodine complex
that is retained better in acidic environments.

2. Cell Wall Theory (Most widely accepted)

• Gram-positive bacteria:
• Have a thick peptidoglycan layer (50–100 layers).
• This dense, cross-linked layer acts as a permeability barrier.
• Alcohol (decolorizer) causes the peptidoglycan to shrink, closing
pores and trapping the dye-iodine complex inside.
• Gram-negative bacteria:
• Have a thin peptidoglycan layer with loose cross-linking.
• Their outer membrane contains lipopolysaccharides that are easily
disrupted by alcohol.
• Decolorizer increases permeability, allowing crystal violet–iodine
complexes to escape.
• After decolorization, these cells take up the counterstain (safranin
or fuchsine) and appear pink/red.
 Ziehl-Neelsen Technique (Hot Method)

1. Smear Preparation
• Use a clean, grease-free, scratch-free glass slide.
• Prepare a smear 2 × 3 cm in size using the yellow purulent portion
of the sputum.
• Smear should be evenly spread — not too thick or too thin.
• A properly prepared smear allows printed text to be seen through
it.
• Conduct smear preparation near a flame (within 6 inches) to reduce
contamination from aerosols by heat coagulation.

2. Heat Fixation
• Air-dry the smear for 15–30 minutes.
• Heat-fix by passing the slide over a flame 3–5 times, for 3–4
seconds each time.
• This step coagulates proteins, fixing the bacteria to the slide.

3. Staining Procedure

Step 1: Primary Stain

• Flood the slide with 1% strong carbol fuchsin for 5 minutes.
• Apply intermittent heating from below until vapors rise (do not
overheat).
• Ensure the stain does not dry:
• Add more stain and reheat if needed.
• Rinse with tap water until all excess stain is washed off.
• At this stage, the smear appears red.

Step 2: Decolorization
• Pour 25% sulfuric acid over the slide for 2–4 minutes.
 • Gently rinse with tap water and tilt the slide to drain.
• If the slide is still red:
• Reapply sulfuric acid for 1–3 minutes, then rinse again.
• Wipe the back of the slide with a swab dipped in sulfuric acid.

Step 3: Counterstain
• Add 0.1% methylene blue to the slide for 30 seconds.
• Rinse gently with tap water and allow to air-dry.

4. Microscopy
• Examine the slide under:
• 40× objective to locate a suitable area.
• Then use 100× oil immersion for detailed observation.

Interpretation
Mycobacterium tuberculosis appears as long slender, straight or slightly
curved and beaded, red colored acid- fast bacillus. Other non-acid fast
organisms present in the smear and the background take up the counter
stain and appear blue.
CULTURE MEDIA
 [Link]/Basal media [Link] media
[Link] Agar
[Link] Water [Link] Agar
[Link] Broth
[Link] Agar

[Link] Media [Link] media

[Link] media [Link] conkey Agar

[Link]
[Link]’s Cooked Meat Broth.
[Link] Heart Infusion.

1. Peptone Water

• Type: Basal (simple) media

• Composition:
• Peptone – 1%
• NaCl – 0.5%
• Distilled water – 100 mL
• Sterilization: Autoclave at 121°C for 15
min.
• Uses:
1. Base for sugar media
2. Used in indole test
NB: Colorless to very pale yellow
2. Nutrient Broth

• Type: Basal (simple) media

• Composition:
• Peptone – 1%
• Beef extract – 0.3%
• NaCl – 0.5%
• Distilled water – 100 mL
• Sterilization: Autoclave at 121°C
for 15 min
• Uses:
1. General purpose growth medium
NB: Light yellow or pale yellow 2. Used for subculturing and
maintenance of stock cultures

3. Nutrient Agar

• Type: Basal (simple solid)

media
• Composition:
• Peptone – 1%
• Beef extract – 0.3%
• NaCl – 0.5%
• Agar – 2%
• Distilled water – 100 mL
• Sterilization: Autoclave at
121°C for 15 minutes
Nutrient Agar – Preferred Uses
1. For performing biochemical tests
• Nutrient agar does not contain any inhibitory substances.
• It supports the growth of many bacteria, making it suitable for
basic biochemical tests like:
• Oxidase test
• Catalase test
• Slide agglutination test
2. For studying colony morphology
• On nutrient agar, bacteria grow freely and show their natural
colony characteristics (size, shape, elevation, surface, edge, opacity).
• This helps in primary identification.
3. For pigment demonstration
• Certain bacteria produce characteristic pigments only on simple
nutrient media.
• Example:
• Pseudomonas aeruginosa green pigment (pyocyanin, fluorescein)
• Serratia marcescens red pigment (prodigiosin)
4. Blood Agar

• Type: Enriched & Differential

• Composition:
• Nutrient agar base
• 5–10% defibrinated sheep blood
(added after autoclaving, at ~45–50°C)

• Sterilization: Base is autoclaved

at 121°C for 15 min; blood added
aseptically afterward

• Uses:
 1. Growth of fastidious organisms (e.g. Streptococcus)
2. Detection of hemolysis (α, β, γ)

Types of Hemolysis
1. Alpha (α) Hemolysis – Partial (Greenish)
• Appearance: Greenish or brownish discoloration around colonies (due
to partial lysis and conversion of Hb methemoglobin).
• Examples:
• Streptococcus pneumoniae
• Viridans streptococci (S. mutans, S. mitis)

2. Beta (β) Hemolysis – Complete (Clear Zone)

• Appearance: Clear, transparent zone around colonies (complete lysis
of RBCs).
• Examples:
• Streptococcus pyogenes (Group A strep)
• Streptococcus agalactiae (Group B strep)
• Listeria monocytogenes
• Staphylococcus aureus

3. Gamma (γ) Hemolysis – None

• Appearance: No hemolysis; agar remains unchanged.
• Examples:
• Enterococcus faecalis
• Non-hemolytic streptococci
5. Chocolate Agar
• Type: Enriched media
• Composition:
• Nutrient agar base
• 5–10% sheep blood (added while
hot to lyse RBCs)
• Sterilization: Same as blood agar;
blood added at 70–80°C to lyse cells
• Uses:
1. Growth of Haemophilus influenzae
and Neisseria species
2. Culturing other highly fastidious
organisms

6. MacConkey Agar
• Type: Selective & Differential &
indicator
• Composition:
• Peptone, bile salts, lactose, neutral
red (indicator), agar (PLANB)
• Crystal violet (to inhibit Gram-
positive bacteria)
• Sterilization: Autoclave at 121°C for
15 min
• Uses:
1. Isolation of Gram-negative enteric
bacilli
2. Differentiates lactose fermenters
(pink colonies) vs. non-lactose fermenters
(colorless)
 [Link] blood agar is enriched and differntial?

• Enriched: Supports fastidious organisms via added blood.

• Differential: Shows visible differences in hemolysis, helping identify
species.

[Link] MacConkey Agar: Selective, Differential, and Indicator?

Selective:
Bile salts & crystal violet inhibit Gram-positive bacteria.
Differential:
Differentiates lactose fermenters vs. non-fermenters.
Indicator:
Neutral red indicates pH change due to acid production from
lactose.

MacConkey Agar – Differential Property

• It contains lactose and a pH indicator (neutral red).
• Based on lactose fermentation, organisms are divided into two
groups:

1. Lactose fermenters (LF):

• They ferment lactose acid is produced pH drops colonies
turn pink/red.
• Example: Escherichia coli.
2. Non-lactose fermenters (NLF):
• They do not ferment lactose no acid colonies remain colorless
or pale.
• Example: Shigella.
 7. Lowenstein-Jensen (LJ) Medium
• Type: Selective enriched medium
(for Mycobacterium)
• Composition:
• Coagulated hen’s egg
• Malachite green (inhibits
contaminants)
• Glycerol, mineral salts
• Sterilization: Inspissation at 85–
90°C for 45 minutes on 3 consecutive
days
• Uses:
1. Isolation of Mycobacterium
tuberculosis
2. Maintains viability of mycobacteria
for longer periods

8.
Robertson’s Cooked Meat (RCM) Medium
• Type: Anaerobic enrichment medium
• Composition:
• Cooked minced meat (horse/ox)
• Nutrient broth
• Sterilization: Autoclave at 121°C
for 15 minutes
• Uses:
1. Growth of anaerobic bacteria (e.g.,
Clostridium)
2. Storage/transport of anaerobes
9. Loeffler’s Serum Slope
• Type: Enriched medium
• Composition:
• Coagulated serum (e.g., horse
serum)
• Glucose
• Nutrient broth
• Sterilization: Inspissation (like LJ
medium)
• Uses:
1. Isolation of Corynebacterium
diphtheriae
2. Demonstration of metachromatic
granules

10. Dorset Egg Medium

• Type: Storage medium
• Composition:
• Hen’s egg
• Saline or nutrient broth
• Sterilization: Inspissation at 85–
90°C
• Uses:
1. Long-term storage of bacterial
cultures (e.g., M. tuberculosis)
2. Transport of sensitive organisms
Why autoclaving is not done for Lowenstein–Jensen (LJ) medium?

• LJ medium is an egg-based medium used for culturing

Mycobacterium tuberculosis.
• The medium contains egg proteins, glycerol, malachite green, potato
flour, etc.

Reason autoclave is not used

1. Egg coagulation:
• Autoclaving at 121°C will coagulate the egg proteins unevenly and
destroy the medium.
• It becomes unsuitable for bacterial growth.
2. Glycerol decomposition:
• Heat of autoclaving may break down glycerol, producing toxic
substances that inhibit growth.
3. Malachite green instability:
• Autoclaving reduces the selective inhibitory property of malachite
green (which suppresses contaminants).

What is done instead?

• Inspissation (coagulation at 80–85°C for 45 min on 3 consecutive
days) is used.
• This coagulates egg proteins uniformly, giving a solid slant without
damaging nutrients.
 Biochemical Identification

After examining colony morphology and doing a Gram stain of a culture

smear, appropriate biochemical tests are selected to identify the
organism.
1. Initial tests for all colonies:
• Catalase test
• Oxidase test
2. For Gram-negative bacilli:
The commonly performed routine tests are remembered as ICUT:
• I – Indole test
• C – Citrate utilization test
• U – Urease hydrolysis test
• T – Triple sugar iron (TSI) test
3. For Gram-positive cocci:
The useful biochemical test is:
• Coagulase test (to identify Staphylococcus aureus).

Additional Biochemical and Susceptibility Tests

• CAMP (Christie-Atkins-Munch-Petersen) test: Used for identification
of Group B Streptococcus.
• Bile esculin hydrolysis test: Used for the identification of
Enterococcus species.
• Heat tolerance test: Another confirmatory test for Enterococcus,
based on their ability to grow at higher temperatures.
• Inulin fermentation test: Used for the identification of
pneumococcus (Streptococcus pneumoniae).
• Bile solubility test: Also used for identification of pneumococcus.
 TEST

[Link] test

Rapid bubbling indicates

catalase-positive.

No or very minimal bubbling

catalase-negative.

[Link] test
Oxidase positive (deep purple)

Oxidase negative (no color

change)

[Link] test

Indole positive: A red colored ring is

formed near the surface of the broth.

Indole negative :Yellow colored ring is

formed near the surface of the broth.
The test is used to check whether certain bacteria can produce an
enzyme called tryptophanase. This enzyme breaks down the amino acid
tryptophan, which is present in the medium, into indole.

After overnight incubation of the bacterial culture in broth, Kovac’s

reagent is added. If indole has been produced, the reagent reacts with it
and forms a cherry red colored ring near the surface of the medium.
• Indole positive: A red-colored ring appears near the surface of the
broth. Examples of bacteria showing this reaction are Escherichia coli,
Proteus vulgaris, and Vibrio cholerae.
• Indole negative: A yellow-colored ring appears near the surface of
the broth. Examples of bacteria showing this reaction are Klebsiella
pneumoniae, Proteus mirabilis, Pseudomonas, and Salmonella.

[Link] test
• Positive: The medium turns blue.

• Negative: The medium remains

green.
Citrate Utilization Test

This test checks whether certain bacteria can use citrate as the only
source of carbon for their growth. If they do, they produce alkaline
metabolic products.

The test is carried out on Simmon’s citrate medium. Bacteria that can
utilize citrate will show visible growth on the medium and cause a color
change from the original green to blue.
• Citrate positive: Klebsiella pneumoniae, Citrobacter, Enterobacter.
• Citrate negative: Escherichia coli, Shigella.

[Link] test

• Positive: The medium turns

bright pink.

• Negative: The medium stays

yellow to light orange

Some bacteria produce the enzyme urease, which breaks down urea in
the medium to release ammonia. This makes the medium alkaline.

The test is performed on Christensen’s urea medium, which contains the

pH indicator phenol red. In an alkaline medium, phenol red changes color
to pink.
• Urease positive: Klebsiella pneumoniae, Proteus species, Helicobacter
pylori, Brucella.
• Urease negative: Escherichia coli, Shigella, Salmonella.

6. Mannitol

Principle

This medium is designed to detect two properties of bacteria:

1. Motility – whether the organism can move through semi-solid agar.
2. Mannitol fermentation – whether the organism can ferment mannitol
and produce acid.

Medium Composition
• Semi-solid agar (0.3–0.5%) allows motile bacteria to move.
• Mannitol carbohydrate source.
• pH indicator (usually phenol red) detects acid production.
Procedure
• Inoculate the medium with a straight stab using a needle.
• Incubate overnight.
• Observe motility and mannitol fermentation.

Interpretation
1. Motility
• Positive: Diffuse/hazy growth spreading away from the stab line.
• Negative: Growth only along the stab line.
2. Mannitol fermentation
• Positive: Medium turns yellow (acid production).
• Negative: Medium remains red/pink (no acid).

Examples
• Motile, Mannitol fermenter: Escherichia coli.
• Non-motile, Mannitol fermenter: Klebsiella pneumoniae.
• Motile, Non-fermenter: Salmonella, Proteus.
• Non-motile, Non-fermenter: Shigella dysenteriae.

7. Glucose
Principle

Some bacteria can ferment glucose to produce acid with or without gas.
• Acid production lowers pH indicator changes color.
• Gas production collected in an inverted Durham’s tube inside the
medium.

Medium
• Peptone water + 1% glucose
• pH indicator: usually Andrade’s indicator or phenol red
• Durham’s tube to detect gas.

Procedure
• Inoculate the medium with the test organism.
• Incubate at 37°C overnight.
• Observe for color change and gas.

Interpretation
1. Acid positive:
• Medium changes from red yellow (phenol red indicator)
• or pale pink (Andrade’s indicator).
2. Gas positive:
• Bubble appears in Durham’s tube.
3. Negative:
• No color change, no gas.

Examples
• Glucose fermenters: Escherichia coli (acid + gas), Enterobacter,
Klebsiella.
• Non-fermenters: Pseudomonas, Alcaligenes
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 SPECIMEN
IDENTIFICATION

BACTERIA MEDIA MOTILITY GRAM STAIN

[Link] Mac Conkey + Gram -ve Bacilli

Klebseilla
Mac Conkey - Gram -ve Bacilli

Proteus Mac Conkey + Gram -ve Bacilli

Pseudomona
Auruginosa Nutrient + Gram -ve Bacilli

Staphylococcus
Nutrient/Blood Gram +ve cocci
Aureus -

Gram +ve Bacilli

Bacillus Subtilis Nutrient/Blood +
 P. S. B.
[Link] Klebsilella Proteus
Aeruginosa Aureus Subtilis

Indole +ve -ve +ve -ve -ve -ve

Citrate -ve +ve +ve +ve -ve +ve

Urease -ve +ve +ve -ve -ve/+ve -ve

Glucose +ve +ve +ve -ve +ve -ve

-ve -ve +ve -ve /+ve

Lactose +ve +ve

-ve +ve -ve

Manintol +ve +ve -ve

+ve +ve +ve

Catalase +ve +ve +ve

Oxidase -ve -ve -ve +ve -ve +ve

Biochemical Characteristics of Bacillus subtilis

• Biochemical Tests:
• Not applicable (not definitive for identification)
• Standard biochemical tests are not sufficient to definitively identify
B. subtilis, as they may overlap with other Bacillus species.

• Further Test – Blood Agar:

• On blood agar, Bacillus subtilis shows β-hemolytic colonies
• This means it causes complete hemolysis of red blood cells, resulting
in a clear zone around colonies.

• Gelatin Liquefaction Test:

• B. subtilis shows uniform gelatin liquefaction
• However, it does not show a fir-tree (inverted pine tree) pattern,
which is characteristic of Clostridium species.

• Growth on Nutrient Agar:

• B. subtilis grows without discoloring the medium
• The colonies are typically dry, rough, and irregular but the
surrounding agar remains unchanged.
Staphylococcus aureus

Biochemical Test
• Not applicable alone for definitive identification.
• General biochemical tests (like catalase, mannitol fermentation) may
support identification, but are not sufficient alone to confirm S. aureus.

Further Test – Coagulase Test

• Gold standard for distinguishing S. aureus (pathogenic) from other
coagulase-negative staphylococci (CoNS), such as S. epidermidis.

Coagulase Test – Principle

• The test uses citrated plasma from human, rabbit, or pig.
• Plasma contains CRF (Coagulase Reacting Factor).
• Coagulase enzyme, produced by S. aureus, reacts with CRF.
• This complex converts fibrinogen fibrin, leading to clot formation
(visible as a gel or clot).

Interpretation of Coagulase Test

• Positive result:
Clot formation observed
Indicates coagulase-positive staphylococcus
Most likely Staphylococcus aureus (pathogenic)
• Negative result:
No clotting observed
Indicates coagulase-negative staphylococcus (CoNS)
Commonly Staphylococcus epidermidis (non-pathogenic or opportunistic)
 Dieases Causes by Above Bacteria
1. Escherichia coli (E. coli)
• Urinary tract infections (UTI) – most common cause
• Gastroenteritis – especially from enterotoxigenic and
enterohemorrhagic strains
• Neonatal meningitis
• Septicemia
• Pneumonia (especially in hospitalized patients)

2. Klebsiella pneumoniae
• Lobar pneumonia – often in alcoholics and diabetics
• Urinary tract infections
• Liver abscess
• Septicemia
• Hospital-acquired infections

3. Proteus species (e.g., Proteus mirabilis)

• Urinary tract infections – associated with stone formation
• Wound infections
• Septicemia
• Pneumonia (less common)

4. Pseudomonas aeruginosa
• Hospital-acquired pneumonia
• Urinary tract infections
• Burn wound infections
• External otitis (swimmer’s ear)
• Sepsis in immunocompromised patients
• Hot tub folliculitis
• Corneal ulcers (contact lens-related)
5. Staphylococcus aureus
• Skin infections – boils, impetigo, cellulitis
• Osteomyelitis
• Septic arthritis
• Pneumonia
• Endocarditis
• Toxic shock syndrome
• Food poisoning
• Wound infections

6. Bacillus subtilis
• Usually non-pathogenic
• Rare cases may include:
• Opportunistic infections in immunocompromised individuals
• Food spoilage-related gastroenteritis (very rare)
Klebsiella – Colony Characteristics on
MacConkey Agar

• Type: Lactose fermenter (LF)

• Appearance: Large, round, convex, pink
colonies
• Surface: Moist, glistening, smooth
• Consistency: Mucoid / sticky (due to
thick polysaccharide capsule)
• Key point: If touched with a loop
colony produces a string (string test
positive).

E. coli – Colony Characteristics on

MacConkey Agar

• Type: Lactose fermenter (LF)

• Appearance: Medium-sized, round,
pink colonies
• Surface: Smooth, moist, non-mucoid
• Color intensity: Often bright pink
(sometimes with a surrounding pink
precipitate zone due to bile salt
precipitation).
• Consistency: Soft, non-sticky (unlike
Klebsiella).
Proteus – Colony Characteristics on
MacConkey Agar

• Type: Non-lactose fermenter (NLF)

• Appearance: Pale, colorless colonies
(no pink color since lactose is not
fermented)
• Swarming: On nutrient agar and
blood agar shows swarming motility;
On MacConkey agar swarming is
inhibited due to bile salts.
• Surface: Colonies appear translucent,
irregular, sometimes rough-edged.
• Consistency: Non-mucoid.

Staphylococcus aureus – Colony Characteristics

on Nutrient Agar

• Size: Medium to large colonies

• Appearance: Circular, convex, smooth
• Color: Golden yellow pigment
(characteristic)
• Opacity: Opaque
• Consistency: Buttery (creamy) in
texture
 Bacillus subtilis – Colony Characteristics on
Nutrient Agar

• Size: Large colonies

• Shape: Irregular margins, spreading
colonies
• Surface: Rough, dry, flat or slightly
wrinkled
• Opacity: Opaque
• Consistency: Leathery, tough (not
soft like staphylococci)
• Color: Dull, whitish to cream colored

Pseudomonas aeruginosa – Colony

Characteristics on Nutrient Agar

• Size: Medium to large colonies

• Shape: Circular, often irregular
edges
• Surface: Smooth or sometimes rough
• Color: Dull opaque colonies, often
with a greenish discoloration of the
medium due to pyocyanin and fluorescein
pigments
• Odor: Characteristic fruity/grape-like
odor
• Consistency: Non-mucoid (except
Dif pigment-
some strains which may be mucoid in
Pyocyanin,Pyoverdine.
cystic fibrosis patients)
Non dif pigment-
Pyorubin,Pyomelanin.
 Serology

Serological tests are very important in diagnosing bacterial infections.

They work by detecting either:
• Antigen (part of the bacteria) or
• Antibody (produced by the patient against the bacteria)

in the patient’s serum, using different immunological methods such as

precipitation, agglutination, ELISA, and rapid tests.

Important Serological Tests for Bacterial Infections

• Widal test used for diagnosis of enteric fever (typhoid)
• Standard agglutination test for brucellosis
• Microscopic agglutination test and rapid diagnostic test for
leptospirosis
• Weil-Felix test for rickettsial infections
• VDRL (Venereal Disease Research Laboratory) test and RPR (Rapid
Plasma Reagin) test for syphilis
• ELISA available for many bacterial diseases such as chlamydial
infections, brucellosis, Mycoplasma pneumonia, leptospirosis, and
rickettsial infections
ASO titer test.

• Rheumatic fever.
• Post-streptococcal glomerulonephritis.

Q. Identify the Test?

Latex agglutination test for ASO titer

Q. Principle of the Test:

Antigen coated on latex particle reacts with specific antibody in
the patient’s serum.
If agglutination occurs, it indicates presence of antibodies (ASO
antibodies in this case).
This is called the latex agglutination test.
the Test -
re

*
Q. Diagram:
E
The diagram shows:
• Positive: visible clumping (agglutination)
• Test: current patient result
• Negative: no clumping

Interpretation:
Test positive indicates ASO titer > 200 IU/mL
 MOTILITY TEST
Hanging Drop Experiment

Purpose / Use:
• To demonstrate bacterial motility (living state).
• To observe true motility vs Brownian movement.
• Also helps to see shape and arrangement of bacteria.

Principle:

Motile bacteria move in different directions due to their flagella, while

non-motile bacteria show only Brownian movement (shaking due to water
molecules).

Procedure:
1. Take a cavity slide (concave depression).
2. Apply a thin ring of vaseline around the cavity.
3. Put a drop of bacterial suspension on a coverslip.
4. Invert the cavity slide over the coverslip so the drop hangs into the
cavity.
5. Examine under microscope.

Observation:
• Motile bacteria: Show directional, darting, zig-zag, or tumbling
movements.
• Non-motile bacteria: Show only Brownian movement (to and fro
vibration, not true movement).
Motility Types & Examples

• Tumbling – Listeria
• Gliding – Mycoplasma
• Shaking – Clostridium
• Darting – Vibrio cholerae
• Swarming – Proteus
• Corkscrew – Spirochete

Some other test for motility

1. Wet mount method

2. Semi-solid agar stab test
2. TTC motility test
4. Swarming motility test
 PPE KITS

Donning (Putting On PPE)

1. Hand Hygiene – Wash hands with soap or use hand sanitizer.
2. Gown – Put on the gown; tie at neck and waist.
3. Mask or Respirator – Secure over nose and mouth; adjust fit.
4. Goggles or Face Shield – Place over eyes or face.
5. Gloves – Wear gloves over gown cuffs.

Tip: Ensure all PPE is secure with no exposed skin.

Doffing (Removing PPE)
1. Gloves – Remove carefully without touching skin.
2. Goggles or Face Shield – Handle only by straps/sides.
3. Gown – Untie and pull away from the body; turn inside out.
4. Mask/Respirator – Remove from behind (avoid touching front).
5. Hand Hygiene – Perform immediately after PPE removal.

Tip: Always avoid touching the front (contaminated side) of PPE.

Advantages of PPE
• Protects healthcare workers from infections and hazardous
materials
• Prevents cross-contamination between patients and staff
• Essential in outbreaks/pandemics (e.g., COVID-19, TB)
• Easy to use with minimal training

Disadvantages of PPE
• Can be uncomfortable and cause heat/stress during long use
• Improper donning/doffing increases risk of contamination
• Limits mobility and visibility (especially with face shields/goggles)
• Costly and generates waste (especially disposable PPE)
HAND HYGENE
 STERILIZATION
VS DISINFECTANT

Sterilization: It is the process by which all living microorganisms

including viable spores, are,destroyed with reduction of at least 106 log
colony forming units (CFU) of microorganisms and their spores.

Disinfection: It refers to a process that destroys or removes most if not

all pathogenic organisms but not bacterial spores with reduction of at
least 103 log CFU of microorganism but not spores.
BIOMEDICAL WASTE
MANAGEMENT

VIVA + OSPE
 INSTRUMENT

1
Two uses of sterile syringe:
.

1. To inject medications or
vaccines (IM, IV, SC, ID routes).
2. To withdraw blood or body
fluids for testing or drainage.

2. Two uses of sterile swab stick:

1. To collect samples (e.g.

throat, wound, nasal) for lab
testing.
2. To clean or disinfect skin
before injections or minor
procedures.

3. Two Uses of an Inoculation Loop:

1. To transfer microorganisms
aseptically
• Used to pick and transfer
bacterial colonies from one medium to
another (e.g., from agar to broth).
2. To perform streaking for isolation
on agar plates
• Helps in spreading bacteria over
the surface to obtain isolated colonies.
Sterilization of Inoculation Loop

Method: Flame Sterilization (Red-heat method).

4. Sterile specimen container.

1. To collect clinical samples
• Such as urine, sputum, stool, pus,
or other body fluids for
microbiological, pathological, or
chemical analysis.
2. To transport specimens to the
laboratory without contamination
• Maintains sample integrity and
prevents growth of unwanted
organisms.
Plastic specimen containers are sterilized by gamma irradiation or EtO
gas; they are supplied pre-sterilized and are disposable.

5. 1. Detection of antibodies against HIV-1

and HIV-2.
2. Differentiation between HIV-1 and HIV
infections.

Sterilization of Nitrocellulose Membrane

• UV irradiation – 15–30 min
(common lab method).
• 70% ethanol – quick dip, air-dry.
• Ethylene oxide gas – industrial.
• Do NOT autoclave or use hot air
oven (heat damages membrane).
6. Two Uses of the Microtiter Plate (shown in
image):
1. Used in ELISA (Enzyme-Linked
Immunosorbent Assay):
• For detecting antigens or antibodies
(e.g., HIV, hepatitis, dengue, etc.)
2. Used in antimicrobial susceptibility
testing:
• Helps determine the Minimum
Inhibitory Concentration (MIC) of
antibiotics against bacteria.

Microtiter plates are sterilized by gamma irradiation/EtO (industrial); in

lab, UV or 70% alcohol is used. Heat methods not suitable for disposable
plastic plates.

7 Uses
. • Smear prep (blood, sputum, stool).
• Staining (Gram, ZN, Giemsa).
• Wet mount, hanging drop.
• Special tests (KOH, agglutination).

Sterilization
1. Hot Air Oven – 160°C × 1 hr
(preferred).
2. Chemical – 5% phenol / 1%
hypochlorite (for used slides before
washing).
3. Flame – quick pass, not routine
(only for fixing smear).
8. Test Tube

Uses
• Culture media preparation (broth,
slant, deep tubes).
• Biochemical tests (sugar
fermentation, citrate, urea test).
• Collection of clinical samples
(urine, sputum, serum).
• Heating reactions (coagulation,
clot retraction).

Sterilization
1. Hot Air Oven – 160°C × 1 hr (for dry, empty glass tubes).
2. Autoclave – 121°C, 15 lb, 15–20 min (for tubes containing media,
fluids).
3. Chemical disinfectant – temporary, for contaminated tubes before
cleaning.

9. Petri Dish

Uses
• Culture of bacteria, fungi on
solid media.
• Antibiotic sensitivity testing
(Kirby–Bauer).
• Colony morphology study.
• Isolation of pure cultures.
• Special tests (CAMP test, etc.).
Sterilization
1. Glass Petri dish
• Hot Air Oven: 160°C × 1 hr (preferred).
• Autoclave: 121°C, 15 lb, 15–20 min (for media-filled plates, not for
empty dry glass).
2. Plastic (disposable) Petri dish
• Not heat-resistant sterilized by Gamma irradiation / Ethylene
oxide at manufacturing.
• Usually used once and discarded.

CULTURE

Blood Culture Media

• Recovery of bacteria from blood is difficult
because:
1. They are usually present in very small
numbers.
2. Many blood pathogens are fastidious
(require special nutrients).
• Therefore, enriched media are used.
• Blood culture media can be of two types:
1. Conventional media
2. Automated media

[Link] Blood Culture Media

Two types:
1. Monophasic medium
• Contains only a liquid phase: Brain Heart Infusion (BHI) broth.
2. Biphasic medium
• Contains two phases:
 • Liquid phase – BHI broth
• Solid phase – BHI agar slope

Identification of Monophasic vs Biphasic Blood Culture Medium

1. Monophasic Medium (only broth)
• Appearance: Single liquid phase only (clear/turbid broth in the
bottle).
• No agar slope or solid part is visible.
• Looks like just a broth medium filling part of the bottle.
2. Biphasic Medium (broth + agar slope)
• Appearance: You will see two distinct phases:
• Liquid phase BHI broth at the bottom.
• Solid phase Agar slope (slanted agar) coating the inside surface
of the bottle above the broth.
• If growth occurs, colonies may be directly visible on the agar slope.
Two Uses of Dorset Egg Medium:
1. Cultivation of Mycobacterium tuberculosis
• Used as a solid medium for growing M.
tuberculosis and other mycobacteria.
2. Storage and maintenance of bacterial
cultures
• Suitable for long-term preservation of
Mycobacterium species without rapid drying or
contamination.
Two Uses of an Anaerobic Blood Culture Bottle:
1. To detect anaerobic bacterial infections in
the blood
• Identifies bacteria that grow without
oxygen, such as Clostridium, Bacteroides, and
Fusobacterium species.
2. To assist in diagnosis of deep tissue or
abscess-related infections
• Useful when infections are suspected
from intra-abdominal, pelvic, or post-surgical
abscesses, where anaerobes are common.

Two Uses of an Aerobic Blood Culture Bottle:

1. To detect aerobic bacterial or fungal
infections in the blood
• Identifies organisms like E. coli,
Staphylococcus aureus, Pseudomonas, etc.,
that need oxygen to grow.
2. To guide appropriate treatment by
antibiotic sensitivity testing
• Once the aerobic organism is isolated, it
helps choose the most effective antibiotic.
 OSPE on Specimen and Microscopic image.
1.
For Hookworm (Ancylostoma duodenale /
Necator americanus):
• Causative organism:
– Ancylostoma duodenale
– Necator americanus
• Route of infection:
– Skin penetration by filariform larva (L3
stage)
• Affected organ:
– Small intestine (jejunum mainly)
• Infective form:
– Filariform larva (L3 stage)

2.
For Taenia (tapeworm infection):
• Causative organism:
– Taenia solium (pork tapeworm)
– Taenia saginata (beef tapeworm)
• Route of infection:
– Ingestion of undercooked pork (T. solium)
or undercooked beef (T. saginata) containing
cysticerci (larval stage).
• Affected organ:
– Small intestine (adult worm lives here).
– In case of T. solium larvae may also
lodge in brain, muscle, eye, subcutaneous
tissue (cysticercosis).
• Infective form:
– Cysticercus cellulosae (larval cyst) in meat.
3.
For Ascaris (roundworm infection):
• Causative organism: Ascaris
lumbricoides
• Route of infection: Ingestion of
embryonated eggs with contaminated food
or water
• Affected organ: Small intestine (larvae
also migrate through liver, heart, lungs
before returning to intestine)
• Infective form: Embryonated egg
(containing larva)

4.
• Causative organism:
• Wuchereria bancrofti (most common,
>90% cases)
• Brugia malayi (less common)
• Route of infection: Bite of infected
female Culex (for W. bancrofti) or Mansonia/
Anopheles (for B. malayi) mosquito.
• Affected organ/system: Lymphatic
system lymph nodes, lymphatic vessels
(leading to lymphatic filariasis, elephantiasis).
• Infective form: Filariform larva (L3
stage) introduced into human skin by mosquito
bite.
• Diagnostic stage: Microfilaria (seen in
peripheral blood smear, usually collected at
night – nocturnal periodicity).
Drawing of Brugia Malayli.

5.

Plasmodium falciparum (Ring stage)

• Causative organism: Plasmodium falciparum
• Route of infection: Bite of infected female Anopheles mosquito
• Affected organ/system: RBCs (blood), later liver (initial schizogony),
systemic circulation causes malaria
• Infective form to human: Sporozoite (injected by mosquito bite)
• Diagnostic form in peripheral smear: Ring form trophozoite (as seen
in the slide)

Characteristic features of P. falciparum ring form in smear

1. Very delicate, thin ring inside RBC.
2. Often multiple rings inside a single RBC (a key differentiating point).
3. Accolé/appliqué form – ring appears at the margin of RBC.
4. Chromatin dot is small and single (sometimes double).
6.

Plasmodium vivax (Trophozoite stage)

• Causative organism: Plasmodium vivax
• Route of infection: Bite of infected female Anopheles mosquito
• Affected organ/system: RBCs (blood) + Liver (latent hypnozoites can
persist in hepatocytes)
• Infective form to human: Sporozoite
• Diagnostic form in peripheral smear: Trophozoite, Schizont,
Gametocyte (depending on stage)

Characteristic features of P. vivax trophozoite in smear

1. RBC enlarged compared to normal.
2. Trophozoite is amoeboid (irregular shape, spreading in cytoplasm).
3. Presence of Schüffner’s dots (stippling) in RBC cytoplasm.
4. Cytoplasm of trophozoite: bluish; chromatin dot: reddish.
5. Seen in benign tertian malaria.
7.

• If you see a neat, oval, thick-walled egg with embryo Fertilized.

• If you see a bigger, elongated, irregular egg with messy granules
and no embryo Unfertilized.

Ascaris lumbricoides
• Causative organism: Ascaris lumbricoides (roundworm)
• Route of infection: Fecal–oral route (ingestion of infective eggs with
contaminated food/water)
• Infective form: Embryonated fertilized egg (NOT unfertilized egg, as
it cannot develop further)
• Affected organ:
• Larval migration: lungs (transient – Loeffler’s syndrome)
• Adult worm: small intestine (jejunum/ileum, occasionally bile duct/
pancreatic duct)

Egg Morphology (seen in stool microscopy)

Fertilized Egg
 • Size: 60 × 45 µm (smaller).
• Shape: Round/oval, regular outline.
• Shell: Thick, triple-layered; outer mammillated albuminous coat,
brown in color.
• Contents: Organized ovum / embryo (can develop if embryonated).
• Infective form when embryonated.

Unfertilized Egg
• Size: 90 × 45 µm (larger).
• Shape: Elongated, irregular outline.
• Shell: Thin, irregular, poorly formed albuminous coat.
• Contents: Disorganized refractile granules, no embryo.
• NOT infective.

Fertilized egg Unfertilised egg

8.

9.
10.
 CLINICAL CASE

1.

Visceral Leishmaniasis (VL)

a) Disease: Visceral Leishmaniasis (VL)

b) Causative Organism and Vector:

• Organism: Leishmania donovani (protozoal parasite)
• Vector: Phlebotomus species (sandfly)

c) Diagnostic Test:
• rK39 Test (also called 2K36 test):
• A specific rapid immunochromatographic test
• Detects antibodies against Leishmania
• Uses a recombinant leishmanial antigen (rK39) consisting of 39
amino acid repeats

d) Complication / Sequela:
• PKDL – Post Kala-azar Dermal Leishmaniasis
• Clinical features:
1. Depigmented or hypopigmented macules over the skin
2. Erythematous patches, sometimes in a butterfly distribution
3. Nodular lesions
2.

V)Post ex pro.

i) Causative Agent:
• Virus: Human Immunodeficiency Virus (HIV)
• Type: RNA virus

ii) Subtype:
• Subtype C – most common worldwide and also the dominant
subtype in India

iii) Serotype:
• Serotype 1

iv) Opportunistic Infections:

• Associated infections:
• Pulmonary tuberculosis (PTB) is a common opportunistic infection
• Chronic infections of various organs may occur due to
immunosuppression

V).HIV Post-Exposure Prophylaxis (PEP) Flowchart

1. Exposure occurs
• Needle-stick, sharp injury, or mucocutaneous exposure
2. Assess source
• HIV-positive PEP indicated
 • Source unknown PEP indicated
• HIV-negative Usually no PEP
3. Start PEP immediately
• Within 2 hours of exposure
4. Recommended regimen (TL+LR):
• Tenofovir + Lamivudine
• Lopinavir + Ritonavir
5. Duration:
• 28 days continuous treatment
6. Hospital policy:
• Nodal center for PEP management
• PEP provided free of cost to employee
7. Follow-up:
• Baseline HIV testing
• Repeat HIV tests at 6 weeks, 3 months, and 6 months
• Monitor for drug toxicity and adherence

[Link]

1. Causative Agent:
• Virus: Rabies virus
• Family: Rhabdoviridae
• Genus: Lyssavirus
• Type: Single-stranded RNA, enveloped virus

2. Transmission:
• Through bite or scratch of a rabid animal (commonly dog, bat, or
other mammals)
• Virus is present in saliva of infected animals
Post-Exposure Prophylaxis (PEP)

1. Immediate Wound Care:

• Wash wound thoroughly with soap and running water for 15 minutes
• Apply antiseptic (povidone-iodine if available)

2. Vaccination:
• Rabies vaccine
ID PEP regimen (2-2-2): 2-site ID vaccine is given on days 0, 3 and 7

3. Rabies Immunoglobulin (RIG):

• Indication: Category III exposure (transdermal bites, scratches, or
licks on broken skin)
• Human RIG (HRIG): 20 IU/kg body weight
• Equine RIG (ERIG): 40 IU/kg body weight
• Administer into and around the wound as much as anatomically
possible; remainder intramuscularly

Tetanus

1. Causative Agent:
• Bacterium: Clostridium tetani
• Type: Gram-positive, spore-forming, obligate anaerobe
• Toxin: Produces tetanospasmin (neurotoxin) causing muscle rigidity
and spasms

2. Transmission:
• Entry via contaminated wounds, cuts, burns, or puncture injuries
• Spores are present in soil, dust, and animal feces
• Not transmitted person-to-person
3. Post-Exposure Prophylaxis (PEP):
 One Health Concept

Definition:
• One Health is an integrated, unifying approach that recognizes the
interconnection between human health, animal health, and the
environment.
• It emphasizes collaboration across multiple sectors to achieve
optimal health outcomes for people, animals, and ecosystems.

Key Components:
1. Human Health:
• Prevention and control of infectious and non-infectious diseases in
humans.
2. Animal Health:
• Monitoring and managing diseases in livestock, pets, and wildlife.
3. Environmental Health:
• Addressing factors like water, soil, air, and climate that affect
disease emergence and spread.

Importance:
• Emerging infectious diseases: Most new human infections are
zoonotic (originating from animals), e.g., COVID-19, rabies, avian
influenza.
• Antimicrobial resistance: Misuse of antibiotics in humans and animals
contributes to resistance.
• Food safety & security: Healthy livestock and crops reduce risk of
foodborne illness.
• Ecosystem balance: Healthy environments prevent disease spillover.

Examples of One Health Applications:

• Rabies control through dog vaccination and human PEP
 • Surveillance of zoonotic influenza in poultry
• Monitoring vector-borne diseases like dengue, malaria, or Lyme
disease