GRAM POSITIVE COCCI ➢ Heavily contaminated specimen:

o MSA (Mannitol Salt Agar), CAN, and

PEA (Phenylethyl Alcohol Agar)
➢ Selective Media:
o 1% Mannitol + 7.5% NaCl MSA (only
halophiles grow)
➢ Selective and Differential media:
o CHROMagar / Chromogenic Agar
➢ Chromogen
o Substrate for enzymes
o Color production

S. epidermidis

STAPHYLOCOCCI
General Characteristics
➢ Aerobic or facultative anaerobic except for S.
saccharolyticus
➢ Incubation:
o 18 – 24 hours
➢ Medium sized, appear creamy, white or rarely
light gold and buttery looking
➢ Some species are considered Beta Hemolytic
➢ Normal inhabitants of the skin and mucous
membrane Cultural Characteristics
➢ Some are halophiles S. aureus S. S. S. lugdunensi
➢ Aureus = aurum = gold epidermidis saprophyticus
➢ S. epidermidis’ old name is S. albus - SBA: round, - Small, - Slightly - Hemolytic
smooth, medium, larger and medium
Microscopic Examination white, sized non- colonies with sized
creamy hemolytic, 50% of strain
➢ Best sample: colonies gray to white producing
o Aspirates - Hemolytic colonies yellow
➢ Optimal Recommendation: zones - Some are pigments
around weakly
o Two swabs for Gram stain culture colonies hemolytic
➢ Stained smears from clinical sample: Remember:
o Early diagnosis and treatment 1. Carbohydrate source
➢ Observations: 2. pH indicator
o Numerous gram-positive, along with a. MSA (phenol red)
polymorphonuclear cells in purulent IDENTIFICATION METHODS
exudates, joint fluids, aspirated
➢ Traditional method of differentiation:
secretions, and other fluids are seen
o Oxidation-Fermentation reactions
when these sites are infected with
➢ Rapid method of differentiation:
staphylococci
o Modified Oxidase Test
Isolation and Identification ▪ detects cytochrome oxidase
➢ Routine Lab Culture Media: SBA (Sheep Blood ▪ What is the substrate? (Sterile
Agar) Cotton Swabs?)

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 ▪ blue (+) o Directly react to fibrinogen sample
o Bound to bacterial cell wall
o Bound coagulase/clumping factor
B. Tube Method: more sensitive method,
confirmatory test
o Detects extracellular/unbound or free
coagulase
o Inoculate tube containing plasma and
incubate at 35˚C
o (+) result:
▪ clot/coagulum formation
after 1-4 hours of incubation

➢ Specific method of Identification:

o S. aureus: Coagulase test
o Differentiate S. aureus and S.
lugdunensis

1. Catalase Test
➢ The enzyme catalase mediates the breakdown
of hydrogen peroxide into oxygen and water
➢ The presence of the enzyme in a bacterial
isolate is evident when a small inoculum is
introduced into hydrogen peroxide, and the
rapid elaboration of oxygen bubbles (gas
formation) occurs

2𝐻2 𝑂2 → 2𝐻2 𝑂 + 𝑂2 (𝑔𝑎𝑠 𝑏𝑢𝑏𝑏𝑙𝑒𝑠)

𝑐𝑎𝑡𝑎𝑙𝑎𝑠𝑒

➢ Agua Oxigenada (hydrogen peroxide)

➢ Positive control: S. aureus
➢ Negative control: E. faecalis

STREPTOCOCCI
➢ Belong to the family Streptococcaceae
➢ Catalase-negative, gram-positive cocci that
are usually arranged in pairs or chains
2. Coagulase test ➢ Appear more elongated than spherical
➢ To appear in chains when grown in broth
➢ Best test for pathogenic S. aureus
cultures
A. Slide Method: screen catalase positive colonies

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 ➢ Most members behave like Facultative
anaerobes
➢ Positive for Carbohydrate fermentation
➢ NOT gas producers
➢ Some species are Capnophiles
Classification of Streptococci
➢ Academic/Bergey’s Classification
o Based on temperature requirement
o 4 groups of bacteria
▪ Pyogenic (37˚C)
[Streptococcus pyogenes]
▪ Viridans (both at 37˚C and
45˚C) [Viridans spp. And
Enterococcus faecalis]
• Upper respiratory
tract
▪ Lactococci (both at 10˚C and
37 ˚C) [Streptococcus lactis]
• Dairy products
▪ Enterococci (10˚C, 37˚C, and
45˚C)
➢ Smith and Brown’s Classification
o Based on hemolytic pattern on BAP
➢ Lancefield Classification
o Based on extraction of C-
carbohydrate from cells walls
o Given a specific sero grouping
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1. Bile Esculin Test
➢ Media: Bile Esculin Agar
o It is a selective and differential
medium
➢ Organisms are capable of growth in the
presence of 4% bile and able to hydrolyze
esculin to esculetin. Esculetin reacts with Fe3+
and forms a dark brown to black precipitate
➢ Esculin + Acid → β-D-glucose + Esculetin
➢ Esculetin + Fe3+ → Dark brown color
➢ Positive:
o Growth and blackening of agar
➢ Negative:
o Growth and no blackening of
medium; no growth
2. CAMP Test
➢ Christie Atkinson Munch Peterson
➢ Media: Blood Agar Media
➢ Certain microorganisms (including group B
Streptococci) produce a diffusible extracellular
hemolytic heat-stable protein (CAMP factor)
that acts synergistically with the beta-lysin of
Staphylococcus aureus to cause enhanced lysis
of RBCs
➢ The group B Streptococci are streaked
perpendicular to a streak of S. aureus on
sheep blood agar
➢ Positive:
o Enhanced hemolysis is indicated by an
ARROW HEAD-SHAPED zone of beta-
hemolysis at the junction of the two
organisms.
➢ Negative:
o No enhancement of hemolysis
 3. Bile Solubility Test ➢ Positive:
➢ Reagent: o Deep blue or violet color in 30
o 2% sodium deoxycholate (bile salt) minutes
solution ➢ Negative:
➢ Bile or a solution of a bile salt (e.g., sodium o Colorless or slightly yellow pink color
desoxycholate) rapidly lyses pneumococcal
colonies. Lysis depends on the presence of an
intracellular autolytic enzyme, amidase. Bile
salts lower the surface tension between the
bacterial cell membrane and the medium,
thus accelerating the organism’s natural
autolytic enzyme which induces clearing of
the culture
➢ Positive Result:
o Suspension CLEARS in tube labelled
test and remains TURBID in control
tube
➢ Negative Result:
o Suspension remains TURBID

4. Salt Tolerance Test

➢ Media:
o Brain-Heart infusion broth (BHI) [Pig]
may be used with the addition of NaCl
and an indicator dye
➢ A high salt concentration thus inhibits a range
of bacteria but allows salt-tolerant organisms
such as Enterococci to grow
➢ Organisms capable of growing in the high
salinity medium, utilize the sugar and release
acid as a byproduct of their metabolism. The
pH drops results in the indicator, bromcresol
purple, to change from purple to yellow.
➢ Positive: Purple to Yellow. Note: Turbidity
alone is an indicative of a positive test
➢ Negative: No turbidity and No color change
after 72 hours of incubation

5. Hippurate Hydrolysis Test

➢ Hippurate is the glycine conjugate of benzoic
acid. When Hippurate is hydrolyzed by an
organism, glycine and benzoic acid are
formed. Glycine is deaminated by the
oxidizing agent ninhydrin, which is reduced
during the process. The end product of
ninhydrin oxidation reacts to form a purple-
colored product

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 NEISSERIA o Infected mother to newborn during
birth
➢ Leading cause of STI
➢ Gram negative intracellular diplococci
➢ On culture: (after 24-48 hours incubation)
o Small
o Tan
o Translucent
o Raised
➢ Requires iron for growth
➢ Glucose fermenter
➢ Principal virulence
o Common pili

Clinical Infections
1. Gonorrhea
➢ Means “flow of seed” or “brothel”
➢ An acute pyogenic infection of non-
ciliated columnar and transitional
epithelium with short incubation 2 – 7
days
FAMILY NEISSERIACEAE
➢ Symptoms:
➢ Obligate anaerobes
o Purulent discharge
➢ Non-motile and non-hemolytic
o Lower abdominal pain
➢ Gram negative diplococci with coffee or
o Dysuria
kidney bean shape
o Vaginal bleeding
➢ On culture:
2. Purulent Urethritis (males) and cervicitis
o Small
(females)
o Gray-white
➢ If untreated, may lead to sterility and
o Opaque
perihepatitis (Fitz-hugh-curtis
o Convex
syndrome)
o Glistening
3. Pharyngitis
➢ Capnophiles
4. Anorectal infection
➢ Carbohydrate fermenters
5. Ophthalmia neonatorum
➢ Oxidase positive
6. Purulent
➢ Catalase positive except..
o N. elongata Laboratory Diagnosis
➢ Natural Habitats: 1. Gram Stain Reaction
o Mucous membrane ➢ Appearance of intracellular gram-
o Urogenital tract negative diplococci is diagnostic
➢ >5PMNs/field but no bacteria = Non
Neisseria gonorrhoeae
gonococcal urethritis (PMN =
➢ Never a normal flora
Polymorphonuclear cells)
➢ Can be found in..
➢ Extracellular gram-negative diplococci
o Urogenital tract
= avirulent form
o Anorectal area
2. Culture (Confirmatory)
o Oropharynx
➢ Thayer Martin Agar
o Conjunctiva
o CAP w/ enrichment
➢ Can be transmitted via..
supplement and antibiotics
o Sexual contact

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 ➢ Modified TMA Carbohydrate Utilization Test
o TMA w/ additional
trimethoprim lactate
➢ Martin Lewis Medium
o Vancomycin
o Colistin
o Trimethoprim
o Anisomycin Differential Tests for Pathogenic Neisseria and M.
➢ New York City Agar catarrhalis
o Vancomycin
o Colistin
o Trimethoprim lactate
o Amphotericin B

3. Oxidase test positive

4. Superoxol test:
➢ 30% hydrogen peroxide
5. Immunologic Tests
➢ Fluorescence Antibody Test
➢ Coagglutination test
➢ Molecular Assays
6. Carbohydrate fermentation/Utilization test
7. Phadebact Test
➢ Used for the definitive identification
of Neisseria gonorrhoeae ONLY
➢ In this test, boiled organisms are
examined by using a 1-min
coagglutination technique

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 ACID FAST STAINING MYCOBACTERIA
ACID FAST STAIN
➢ Differential stain which distinguishes
organisms with waxy cell walls that can resist
decolorization with acid alcohol
➢ Acid-fast bacteria have a waxy substance
called mycolic acid (resists decolorization) in
their cell walls which makes them
impermeable to any staining procedures

Notable Acid-Fast Structures

➢ All Mycobacteria-M. tuberculosis, M. leprae,
M. smegmatis and atypical Mycobacterium
➢ Actinomyces
➢ Head of Sperm
➢ Bacterial Spores
➢ Cellular inclusions
➢ Some parasites and cysts

Note:
Mycobacterium tuberculosis
Principle
➢ Slow grower
➢ Carbol Fuchsin is the primary stain in the
➢ Most virulent
procedure, and it contains phenol to help
➢ X, V, Y form
solubilize the cell wall
➢ Slender looking/beaded rods
➢ Heat is also applied during the primary stain
➢ Clinically important
to increase stain penetration. All cell types will
➢ “Cauliflower”
take up the primary stain (Kinyoun method)
➢ Catalase positive
[take note]
➢ Reduce nitrate to nitrite
➢ The cells are then decolorized with acid-
alcohol which decolorizes al cells except the Laboratory Diagnosis
acid-fast ones ➢ Specimen
➢ Methylene blue is then applied to ➢ Sputum
counterstain (Zeihl-Neelsen) o Early morning (3 consecutive days)
o 5-10 mL expectorated sample
o <10 EC and >25 pus cells
➢ Gastric lavage Overnight fasting
o 3 specimen after 3 days
o 20-25 mL gastric secretion
o Do an overnight fasting
➢ Specimen
o Urine
▪ First morning and midstream
catch (3 cons. Days)
▪ 15 mL of urine
o Bronchoscopy specimen

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 ▪ BAL and bronchial washing
▪ Specimen of choice for Non-
Tuberculous Mycobacterium
(NTM)
Culture Media – Solid
➢ Incubation: 35°C (temperature requirement)
in the dark with 5-10% CO2
➢ 30-32°C: M. marinum and M. ulcerans
➢ 42°С: М. хепорі
➢ Tube media: incubated slanted with loose cap
➢ Must be examined weekly
o 3 – 6 weeks to appear for isolates
o Slow growers
➢ Malachite green
o prevent non-mycobacteria
Culture Media – Liquid/Broth
➢ BACTEC 12B and BACTEC 13B
➢ Improves isolation rate of mycobacteria and
reduce recovery time
➢ Reading is about 4 days after inoculation
Staining
➢ General
o 2 of the first 3 sample must be
positive to confirm diagnosis
o 5000 – 10,000 organisms/mL is
needed for positive results
o AFB staining method
▪ Ziehl-Neelsen
▪ Kinyoun

Egg Based Media

➢ Fresh whole eggs, potato flour, glycerol, milk,
potato and malachite green
o Lowenstein Jensen Agar
▪ Most important
▪ Commonly used for
mycobacterium

o Petragani medium
▪ Used to isolate
mycobacterium from heavily
contaminated
o Wallenstein medium
▪ Mycobacterium Avium
Complex (MAC)

Serum Agar-Based Medium

➢ Vitamins, salt, cofactors, glycerol, malachite
green, agar, oleic acid, bovine albumin,
glucose and beef catalase
➢ Middlebrook 7H10-11 Mycobacterium tuberculosis (Koch bacillus)
➢ Mitchison's medium 7H11

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 Number of Number of AFB Report
AFB* seen seen
Fuchsin Stain Fluorochrome
(1000x stain (450x
Magnification) Magnification)
0 0 No AFB seen
1 – 2/ 300 1 – 2/ 70 fields Doubtful;
fields request
another
specimen
1 – 9/ 100 1 – 9/ 50 fields 1+
fields
1 – 9/ 10 fields 4 – 36/ 10 2+
fields
1 – 9/ field 4 – 36/ field 3+
> 9/ fields >36/ field 4+

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 BLOOD CULTURE o Wash hands with soap and water with
➢ A blood culture is a test designed to detect if friction for 15 seconds or use alcohol-
microorganisms such as bacteria and fungi are based hand rub
present in blood • Step 4: Pre Cap
➢ Blood cultures are ordered as a se t, which o Preparing the rubber cap of the bottle
consist of 2 bottles o Wipe the rubber cap with alcohol pad
o 1 for aerobic; 1 for anaerobic in circular motion
o 1 set in 1 arm (2 bottles) • Step 5: Prep the Puncture Site
o 1 set in other arm (2 bottles) o Clean site with chlorhexidine
o Scrub with a back-and-forth motion
Contaminated blood culture
using friction for 30 seconds on dry
➢ If the skin is not adequately cleansed before
skin or 2 minutes on wet skin
drawing blood for culture, bacteria on the skin
o Do not wipe site after cleansing the
will be injected into the bottle, producing a
skin with chlorhexidine
false positive blood culture
• Step 6: Apply Gloves
Principle for Collection • Step 7: Draw Blood
➢ Gloves will be worn in accordance with • Step 8: Mix
standard precautions o Gentle rotation
➢ A physician's order/request must be obtained o Do not shake vigorously
for specimen collection • Step 9 and 10: Label and Send
➢ Appropriate verification of the patient's • Step 11: Document
identity • Step 12: Second Set
➢ Cultures should be drawn before o If 2 sets of blood cultures have been
administration of antibiotics, if possible (nurse ordered, obtain the second set in the
or doctor) same manner as the first, making a
➢ If at all possible, blood cultures should not be new venipuncture at different site
drawn from lines (no IV lines)
Do’s Don’ts
Materials needed Blood culture bottles Do not blow on site, fan
• Venipuncture Set should be held at room or wipe off the solution,
• Sterile Gloves temperature and sent it this may result in
without delay to the contamination of blood
• 2 syringes (adult: 20cc, pediatric: 5cc) [for 2
laboratory within 2 culture
bottles or tubes]
hours
o If there is only 1 syringe used, 1st
Always obtain using Blood culture should not
dispense for aerobic, 2nd for anaerobic strict aseptic technique be drawn through
• 2 needles (adult: 22 gauge or preferable larger to prevent indwelling intravenous
butterfly or standard needle; pediatric: 25- or contamination or intra-arterial catheter
23-gauge butterfly or standard needle unless physician's
• Chlorhexidine swabs (moistened with) request
• Plastic zip lock specimen bags Fill bottles with proper Do not cover bottle bar
volume code and should not
Procedure write over the bar code
• Step 1-3: CHECK, EXPLAIN, WASH On the bottle, write Do not delay the sample
o Identify the patient by checking the MedTech initials, time
arm band or area specific procedure collection, and site
o Explain the procedure to the patient collected
After thoroughly mixing
contents by gently
inverting the bottles,

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 place lab bar code label
vertically on the bottle

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