Part I: Bacterial Culture and • Support the growth of a wide

Inoculation variety of nonfastidious

organisms.
• Examples: Nutrient agar, Tryptic
soy agar.
Introduction • Used as baseline media for many
procedures.
Bacterial culture and inoculation are the
foundation of diagnostic microbiology. 1.2 Enriched Media
Before biochemical testing or
antimicrobial susceptibility testing can • Contain additional growth factors
be done, the organism must first be such as blood, serum, or special
successfully isolated and grown in the nutrients.
laboratory. Culture allows us to: • Examples:
o Blood agar: detects
• Recover organisms from clinical hemolysis (alpha, beta,
specimens. gamma).
• Differentiate contaminants from o Chocolate agar: prepared
pathogens. by heating blood agar to
• Observe colony morphology, lyse RBCs, releasing NAD
pigmentation, and hemolytic (factor V) and hemin
activity. (factor X). Supports
• Provide pure colonies for further Neisseria and
identification and susceptibility Haemophilus.
testing. • Importance: Used for fastidious
organisms requiring extra
nutrients.

Culture methods vary depending on the 1.3 Selective Media

nutritional requirements of the organism,
the type of specimen, and the suspected • Contain inhibitory substances that
pathogen. suppress unwanted organisms
while allowing the growth of the
desired group.
• Examples:
1. Culture Media o Mannitol Salt Agar (MSA):
high salt selects
Culture media provide the nutrients and Staphylococcus. S. aureus
environmental conditions required for ferments mannitol (yellow
bacterial growth. They are categorized colonies).
based on their function: o CNA (Colistin-Nalidixic
Acid) Agar or PEA
(Phenylethyl Alcohol)
Agar: inhibit Gram-
1.1 General Purpose Media negative bacilli, selective
for Gram-positive cocci.
 o MacConkey Agar: bile • Purpose: To isolate individual
salts and crystal violet colonies from a mixed specimen.
inhibit Gram-positives; • Procedure: Inoculating loop
lactose fermentation streaks specimen across agar
differentiates surface in four quadrants,
Enterobacteriaceae. thinning out bacteria until isolated
colonies appear.
1.4 Differential Media • Application: Standard for routine
isolation from clinical samples.
• Distinguish bacteria based on
metabolic reactions.
• Examples:
o Blood agar: hemolytic 2.2 Spread Plate Method
reactions.
o MacConkey agar: lactose • Purpose: To evenly distribute
fermenters (pink colonies) diluted specimen across the
vs non-fermenters surface of agar.
(colorless). • Procedure: A small volume of
sample is pipetted onto the agar
1.5 Enrichment Media and spread with a sterile
spreader.
• Enhance growth of a particular • Application: Useful in quantitative
organism from a mixed specimen. culture and antibiotic assays.
• Examples:
o Selenite broth: enriches
Salmonella.
o LIM broth: used for Group 2.3 Pour Plate Method
B Streptococcus; contains
antibiotics to suppress • Purpose: To grow bacteria within
other bacteria. the agar matrix.
o Todd-Hewitt broth: • Procedure: Diluted specimen is
supports Streptococcus mixed with molten agar and
agalactiae, often used in poured into a Petri dish. Colonies
prenatal screening. grow both on the surface and
inside the agar.
• Application: Useful for counting
viable bacteria.
2. Methods of Inoculation

Correct inoculation techniques ensure

that cultures yield useful, interpretable 2.4 Stab Inoculation
results.
• Purpose: To test oxygen
requirements and motility.
• Procedure: Needle is stabbed
2.1 Streak Plate Method into agar medium. Aerobic
 bacteria grow on the surface, Colony appearance provides important
anaerobes deeper inside. Motile preliminary information. Key features to
organisms spread out from the note:
stab line.
• Application: Commonly used with • Size: pinpoint, small, medium,
motility test media and TSI agar. large.
• Shape: round, irregular,
filamentous.
• Elevation: flat, raised, convex,
3. Incubation Conditions umbonate.
• Margin: entire, undulate, lobate,
filamentous.
• Color: golden yellow (S. aureus),
Once inoculated, cultures require greenish sheen (Pseudomonas),
appropriate incubation conditions: mucoid (Klebsiella).
• Hemolysis (on blood agar):
• Temperature: Most pathogens o Alpha: partial hemolysis,
grow best at 35–37 °C. Some green zone (S.
environmental organisms may pneumoniae).
prefer lower (25–30 °C). o Beta: complete hemolysis,
• Atmosphere: clear zone (S. pyogenes).
o Aerobic: Standard o Gamma: no hemolysis
incubator with ambient air. (Enterococcus).
o Anaerobic: Anaerobe jar,
chamber, or gas- .
generating sachets. Used
for Clostridium,
Bacteroides.
o Capnophilic: 5–10% CO₂ Part II: Biochemical Testing
required (e.g., Neisseria,
Haemophilus).
o Microaerophilic: Reduced
oxygen, increased CO₂ Biochemical tests are the backbone of
(e.g., Campylobacter). bacterial identification. They allow us to
• Time: Most bacteria grow within characterize bacteria based on their
18–24 hrs, but some require enzymatic activities, fermentation
longer (e.g., Mycobacterium). abilities, and metabolic products. No
single test is sufficient; rather, a
combination of tests creates a metabolic
profile that can pinpoint the organism.
4. Colony Morphology

1. General Screening Tests

1.1 Catalase Test • Procedure: Organism applied to
Microdase disk; presence of
• Principle: Detects the enzyme oxidase enzyme produces color
catalase, which breaks down change.
hydrogen peroxide into water and • Results: Blue-purple = positive.
oxygen. • QC Organisms: Micrococcus
• Procedure: Place a drop of 3% luteus (+), Staphylococcus
H₂O₂ on a glass slide, mix with aureus (–).
bacterial colony. • Use: Differentiates Micrococcus
• Results: (+) from Staphylococcus (–).
o Positive: bubbling (release
of O₂).
o Negative: no bubbles.
• QC Organisms: Staphylococcus 2. Carbohydrate Fermentation Tests
aureus (+), Streptococcus
pyogenes (–). • Principle: Many bacteria ferment
• Importance: Differentiates sugars to produce acid,
Staphylococcus (positive) from sometimes gas.
Streptococcus (negative). • Media: Peptone water with a
specific carbohydrate, pH
indicator (phenol red), and
Durham tube.
1.2 Oxidase Test • Procedure: Inoculate and
incubate at 35–37 °C for 24 hrs.
• Principle: Detects cytochrome c • Results:
oxidase enzyme in the electron o Yellow = acid.
transport chain. o Gas bubble in Durham
• Procedure: Smear colony on tube = gas production.
oxidase strip or reagent-soaked o Red = no fermentation.
filter paper (tetramethyl-p- • QC Organisms: E. coli (glucose
phenylenediamine). and lactose positive),
• Results: Pseudomonas (negative).
o Positive: purple within 10–
30 seconds.
o Negative: no color change.
• QC Organisms: Pseudomonas 3. IMViC Tests
aeruginosa (+), E. coli (–).
• Limitations: Old colonies may
give false negatives.
Used primarily to differentiate
Enterobacteriaceae.

1.3 Microdase Test

• Principle: A modified oxidase test 3.1 Indole Test

for Gram-positive cocci.
 • Principle: Detects breakdown of • Principle: Urease enzyme
tryptophan to indole. hydrolyzes urea → ammonia +
• Procedure: Grow bacteria in CO₂, raising pH.
tryptone broth, add Kovac’s • Medium: Urea broth or
reagent. Christensen’s urea agar.
• Results: Red ring = positive. • Results: Pink (alkaline) = positive.
• QC: E. coli (+), Klebsiella • QC: Proteus vulgaris (+), E. coli
pneumoniae (–). (–).

3.2 Methyl Red Test (MR) 4.2 Gelatin Hydrolysis

• Principle: Detects strong acid • Principle: Detects gelatinase

production from glucose enzyme.
fermentation. • Procedure: Inoculate gelatin deep
• Procedure: Grow in MRVP broth, tubes, refrigerate after incubation.
add methyl red indicator. • Results: Liquefaction = positive.
• Results: Red = positive. • QC: Proteus (+), E. coli (–).
• QC: E. coli (+), Enterobacter
cloacae (–).

3.3 Voges-Proskauer (VP) 4.3 Starch Hydrolysis

• Principle: Detects acetoin (neutral • Principle: Detects amylase

end-product) from glucose activity.
fermentation. • Procedure: Grow on starch agar,
• Procedure: Add α-naphthol and flood with iodine.
KOH to MRVP broth. • Results: Clear zone around
• Results: Red = positive. colonies = positive.
• QC: Enterobacter cloacae (+), E. • QC: Bacillus subtilis (+), E. coli (–
coli (–). ).

3.4 Citrate Utilization 4.4 Casein Hydrolysis

• Principle: Tests if organism can • Principle: Protease enzymes

use citrate as sole carbon source. hydrolyze casein in milk agar.
• Procedure: Inoculate Simmons • Results: Clear halo = positive.
citrate agar slant. • QC: Bacillus subtilis (+), E. coli (–
• Results: Blue color = positive. ).
• QC: Klebsiella pneumoniae (+),
E. coli (–). 4.5 Lipid Hydrolysis

• Principle: Lipase enzyme

hydrolyzes fats.
4. Enzyme-Based Hydrolysis Tests • Media: Tributyrin agar.
• Results: Clear halo = positive.
4.1 Urease Test • QC: Staphylococcus aureus (+).
 • Procedure: Stab semisolid agar.
• Results: Diffuse growth = motile.
5. Multi-Reaction Tests • QC: E. coli (+), Klebsiella (–).

5.1 Triple Sugar Iron (TSI) Agar 6.3 PYR Test

• Principle: Differentiates based on • Principle: Detects pyrrolidonyl

glucose, lactose, sucrose arylamidase.
fermentation, H₂S, and gas. • Results: Red = positive.
• Procedure: Stab butt and streak • QC: S. pyogenes (+), S.
slant. agalactiae (–).
• Interpretation:
o Red slant/yellow butt = 6.4 LAP Test
glucose only.
o Yellow slant/yellow butt = • Principle: Detects leucine
glucose + lactose/sucrose. aminopeptidase.
o Black = H₂S. • Results: Red = positive.
o Cracks/bubbles = gas. • QC: Enterococcus faecalis (+),
• QC: E. coli (A/A gas), Salmonella Aerococcus (–).
(K/A + H₂S).
6.5 Bile Esculin Test
5.2 SIM Medium
• Principle: Organism hydrolyzes
• Sulfur: H₂S (black). esculin in bile salts.
• Indole: red after Kovac’s. • Results: Black medium = positive.
• Motility: diffuse growth. • QC: Enterococcus (+), S.
• QC: E. coli (+ indole, motility), pyogenes (–).
Klebsiella pneumoniae (–
motility). 6.6 Growth in 6.5% NaCl

• Principle: Salt tolerance.

• Results: Visible turbidity =
6. Other Key Tests positive.
• QC: Enterococcus (+), S. bovis (–
6.1 Nitrate Reduction ).

• Principle: Detects nitrate → nitrite 6.7 CAMP Test

or nitrogen gas.
• Results: Red after reagents = • Principle: CAMP factor enhances
positive. S. aureus beta-hemolysin.
• QC: E. coli (+), Acinetobacter (–). • Results: Arrowhead hemolysis =
positive.
6.2 Motility Test • QC: S. agalactiae (+), S.
pyogenes (–).
• Principle: Detects flagella-
mediated motility.
6.8 Hippurate Hydrolysis specific bacterial isolate. By providing
information on resistance and sensitivity,
• Principle: Detects glycine from AST guides clinicians in selecting the
hippurate hydrolysis. most appropriate therapy. It also plays a
• Results: Deep purple = positive. key role in monitoring resistance trends
• QC: S. agalactiae (+), S. such as MRSA, ESBL, and VRE.
pyogenes (–).

6.9 ONPG Test

1. Goals of AST
• Principle: Detects β-
galactosidase in late lactose • Identify the most effective
fermenters. antibiotic for treatment.
• Results: Yellow = positive. • Detect emerging resistance
• QC: E. coli (+), Salmonella (–). mechanisms.
• Aid in hospital infection control.
6.10 Decarboxylase Tests • Support surveillance of
antimicrobial resistance globally.
• Principle: Detects amino acid
decarboxylases (lysine, ornithine,
arginine).
• Results: Purple = positive. 2. Factors Affecting AST
• QC: Enterobacter (+ for
ornithine), Klebsiella (–). Several variables can affect accuracy of
results:
6.11 Phenylalanine Deaminase
• Inoculum density (standardized to
• Principle: Detects deamination of McFarland 0.5).
phenylalanine. • Agar depth (must be 4 mm in disk
• Procedure: Add ferric chloride diffusion).
after incubation. • pH of medium (Mueller-Hinton
• Results: Green = positive. agar pH 7.2–7.4).
• QC: Proteus vulgaris (+), E. coli • Incubation temperature and time
(–). (35 °C, 16–20 hrs).
• Antibiotic disk potency (must be
stored and used correctly).
Part III: Antimicrobial Susceptibility
Testing (AST)

3. Conventional AST Methods

Introduction
3.1 Disk Diffusion (Kirby-Bauer
Antimicrobial susceptibility testing (AST) Method)
is an essential part of clinical
bacteriology. It determines which
antibiotics are effective against a
 • Principle: Antibiotic diffuses from o Incubate overnight.
disk into agar, inhibiting growth of o MIC = lowest
susceptible organisms. concentration without
• Procedure: visible growth.
1. Prepare bacterial • Types:
suspension equivalent to o Macrodilution: Tubes with
0.5 McFarland standard. larger volumes.
2. Inoculate entire surface of o Microdilution: 96-well
Mueller-Hinton agar plate. plates, commonly used in
3. Place antibiotic disks on clinical labs.
agar with sterile • MBC (Minimum Bactericidal
forceps/dispenser. Concentration): Subculture clear
4. Incubate at 35 °C for 16– wells onto antibiotic-free agar to
18 hrs. find lowest concentration that kills
5. Measure diameter of 99.9% of bacteria.
inhibition zones in • QC Organisms: Same as disk
millimeters. diffusion.
• Interpretation: Compare zone • Advantages: Quantitative (gives
sizes to CLSI or EUCAST exact MIC).
standards to classify as: • Disadvantages: More labor-
o Susceptible (S) intensive than disk diffusion.
o Intermediate (I)
o Resistant (R)
• QC Organisms:
o E. coli ATCC 25922 3.3 Agar Dilution
o Pseudomonas aeruginosa
ATCC 27853 • Principle: Antibiotic incorporated
o S. aureus ATCC 25923 into agar at known
• Limitations: Not suitable for slow- concentrations.
growing or fastidious organisms • Procedure: Spot inoculate
(e.g., Neisseria gonorrhoeae, multiple organisms on antibiotic-
Haemophilus influenzae). containing agar plates.
• Results: MIC = lowest
concentration plate with no
growth.
3.2 Broth Dilution • Advantages: Reliable for many
organisms at once.
• Principle: Determines the • Disadvantages: Labor-intensive,
Minimum Inhibitory Concentration not used routinely in clinical labs.
(MIC).
• Procedure:
o Serial twofold dilutions of
antibiotic in broth tubes or 3.4 Gradient Diffusion (E-test)
wells.
o Add standardized
inoculum.
 • Principle: Uses a plastic strip 5.1 Detection of MRSA
impregnated with a gradient of
antibiotic concentration. • Cefoxitin disk screen: Preferred
• Procedure: Place E-test strip on over oxacillin for detecting mecA-
inoculated agar. mediated resistance.
• Result: Ellipse of inhibition forms; • mecA gene PCR: Molecular
MIC = where ellipse intersects confirmation.
strip.
• Advantages: Simple, gives
precise MIC.
• Disadvantages: More expensive 5.2 Vancomycin Resistance in
than disks. Enterococci (VRE)

• Vancomycin screen agar: Growth

at ≥6 µg/mL vancomycin
4. Automated AST Systems indicates resistance.
• E-test can also be used.
Modern microbiology labs use
automated systems for faster and
standardized results.
5.3 D-test
• Vitek 2 (bioMérieux)
• Phoenix (BD) • Detects inducible clindamycin
• MicroScan (Beckman Coulter) resistance in Staphylococcus
• Sensititre (Thermo Scientific) aureus.
• Procedure: Place erythromycin
Advantages: and clindamycin disks 15–20 mm
apart. Flattened “D-shaped” zone
• Rapid results (6–12 hrs). near clindamycin = positive.
• Can process large numbers of
samples. 5.4 High-Level Aminoglycoside
• Provides MIC values and Resistance
resistance detection.
• For Enterococcus. Growth in
Limitations: gentamicin or streptomycin high-
level screen agar indicates
• Costly instruments and resistance.
consumables.
• Occasional discrepancies with
reference methods.
6. Quality Control in AST

• Use of standard reference strains

5. Supplemental and Specialized for every batch (e.g., E. coli
Tests ATCC 25922).
 • Media pH and depth checked Gram-positive cocci are among the most
regularly. clinically significant bacteria. They
• Antibiotic disks stored at –20 °C include Staphylococcus, Streptococcus,
or refrigerated. and Enterococcus, which are
• Turbidity standard (0.5 responsible for a wide range of
McFarland) prepared with barium infections, from skin diseases to life-
sulfate or commercial standards. threatening systemic infections.

The catalase test is the primary tool

used to separate these groups:
7. Interpretation and Clinical
Relevance • Catalase-positive:
Staphylococcus, Micrococcus.
• MIC values are correlated with • Catalase-negative:
achievable drug levels in Streptococcus, Enterococcus.
blood/tissues.
• Susceptible (S): Infection likely
responds to therapy.
• Intermediate (I): Response 1. Staphylococcus
uncertain; may work in high
doses or specific sites. 1.1 General Features
• Resistant (R): Treatment failure
expected at normal doses. • Gram-positive cocci in clusters
(grape-like).
• Catalase-positive, facultative
anaerobes.
AST results must be interpreted • Grow well on non-selective
alongside clinical condition and media; tolerate high salt (7.5%).
pharmacology of the drug. • Produce golden/yellow colonies
(especially S. aureus).
Perfect. Let’s now finish your reviewer
with Part IV: Gram-Positive Cocci. This 1.2 Clinically Important Species
will be a long, detailed section (6–7
pages once in PDF) because it covers • Staphylococcus aureus
classification, diagnostic tests, and o Coagulase positive.
clinical relevance. o Ferments mannitol (MSA
yellow).
o Beta-hemolytic on blood
agar.
Part IV: Gram-Positive Cocci o Diseases: skin infections,
food poisoning,
pneumonia, endocarditis,
toxic shock.
Introduction • Staphylococcus epidermidis
o Coagulase negative
(CoNS).
 o Normal skin flora. • Gamma hemolysis (none): No
o Opportunistic (catheter, change.
prosthetic valve o Example: Enterococcus.
infections).
• Staphylococcus saprophyticus 2.3 Lancefield Classification
o Coagulase negative.
o Novobiocin resistant (key • Groups A–H, K–V, based on cell
for differentiation). wall carbohydrate antigens.
o Causes UTIs in young • Clinically important: Group A (S.
women. pyogenes), Group B (S.
agalactiae), Group D
1.3 Key Diagnostic Tests (Enterococcus and non-
enterococcal streptococci).
• Catalase: +
• Coagulase: + in S. aureus only.
• Mannitol fermentation: + in S.
aureus. 3. Major Streptococcal Groups
• Novobiocin sensitivity:
o S. epidermidis: sensitive. 3.1 Group A Streptococcus (
o S. saprophyticus: resistant.
S. pyogenes

)
2. Streptococcus
• Beta hemolytic.
2.1 General Features • Key tests:
o Bacitracin sensitivity:
• Gram-positive cocci in chains or positive.
pairs. o PYR test: positive.
• Catalase-negative. • Diseases: Pharyngitis, scarlet
• Require enriched media (blood fever, rheumatic fever,
agar, chocolate). glomerulonephritis, necrotizing
fasciitis.
2.2 Hemolysis Patterns on Blood
Agar

• Beta hemolysis (complete): Clear 3.2 Group B Streptococcus ( S.

zone around colonies. agalactiae)
o Example: S. pyogenes
(Group A), S. agalactiae • Beta hemolytic.
(Group B). • Key tests:
• Alpha hemolysis (partial): o CAMP test: positive
Greenish discoloration. (arrowhead hemolysis with
o Example: S. pneumoniae, S. aureus).
viridans streptococci. o Hippurate hydrolysis:
positive.
 • Diseases: Neonatal sepsis, o Optochin sensitivity:
meningitis, infections in pregnant positive.
women. o Bile solubility: soluble.
• Diseases: Pneumonia,
meningitis, otitis media, sinusitis.
• Vaccines available for prevention
3.3 Group D and Enterococcus (pneumococcal polysaccharide,
conjugate vaccines).
• Includes Enterococcus faecalis,
Enterococcus faecium.
• Formerly classified as Group D
streptococci. 4. Differentiation Table for Gram-
• Key tests: Positive Cocci
o Bile esculin: positive
(black).
o Growth in 6.5% NaCl:
positive. 5. Clinical Relevance
o PYR test: positive.
• Diseases: UTIs, endocarditis, • Staphylococcus aureus: causes
intra-abdominal infections. skin infections, food poisoning,
• Resistance: Vancomycin- pneumonia, toxic shock. MRSA
resistant enterococci (VRE) are strains complicate treatment.
major hospital pathogens. • Coagulase-negative
staphylococci: associated with
prosthetic devices and catheter
infections.
3.4 Viridans Streptococci • Streptococcus pyogenes:
pharyngitis, rheumatic fever,
• Alpha hemolytic, catalase necrotizing fasciitis.
negative. • Streptococcus agalactiae:
• Includes S. mitis, S. sanguinis, S. neonatal sepsis, infections in
mutans. pregnancy.
• Key features: • Viridans streptococci: dental
o Optochin resistant. caries, endocarditis.
o Bile insoluble. • Streptococcus pneumoniae:
• Diseases: Dental caries, pneumonia, meningitis, otitis
subacute bacterial endocarditis. media; vaccines available.
• Enterococcus: common in
hospital-acquired infections; VRE
is a major concern.
3.5 Streptococcus pneumoniae

• Alpha hemolytic, lancet-shaped

diplococci.
• Key tests: