MICROBIOLOGY PHASE II INTERVIEW

CULTURE
1. What are the normal microbiota of the throat? The pathogens in the throat?
● The normal microbiota of the throat include Alpha hemolytic Streptococcus and
Staphylococcus spp. Streptococcus pyogenes is the most common pathogen of the throat
which is a group A beta-hemolytic Streptococcus (GABHS), and the infection is
commonly known as strep throat or streptococcal sore throat.
○ Streptococcus pyogenes – gram-positive cocci, beta-hemolytic pattern

2. What is the normal microbiota of the urine? the pathogens in the urine?
● The normal microbiota of the urine comes from the presence of contaminating bacteria
from the urinary tract. Most common organisms to grow in the urine are Staphylococcus,
Streptococcus, Lactobacillus, Diphtheroids, Candida.
Pathogens present in urine (give 3):
● Streptococcus pyogenes – gram-positive cocci, beta-hemolytic pattern, catalase negative,
coagulase negative
● Staphylococcus aureus – gram-positive cocci, beta hemolytic pattern, coagulase positive,
catalase positive
○ Coagulase test – use to determine staphylococcus aureus among other
staphylococcus
○ Catalase test – used to determine STREP from STAPH
● Escherichia coli – gram negative bacilli green metallic sheen in EMB
Non-pathogenic in urine (give 3):
● Diphtheroid – bacilli
● Lactobacillus – bacilli
● Staphylococcus epidermidis – gram-positive cocci

3. What are the media used in cervical, urethral & vaginal discharge?
● Routinely, the cultures used in the laboratory for CVUD are BAP, CAP, and MacConkey
Agar but MSA and EMB can also be used if available to further identify and narrow down
the microorganism present.

4. How do you culture cervical, urethral & vaginal discharge?

● To culture cervical, urethral and vaginal discharge, specimens are obtained by either
collecting the discharge or inserting a urogenital swab until the swab is saturated with
exudates then placed in a general enriched medium. In collecting the specimen, calcium
alginate and cotton swabs are not recommended. Dacryon, rayon, and polyester tipped
swab should be used instead.

5. What are the media used in stool culture?

Selective Media Commonly Used to Recover Enteropathogen
CULTURE MEDIA PURPOSE PATHOGENS

Mac Conkey agar To recover Salmonella, shigella (NLF)

Enterobacteriaceae and other
fastidious Gram negative
organisms

Hektoen enteric agar (HE) A highly selective media to Salmonella: blue green with
recover Salmonella, Shigella, black centre

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 contain indicator to detect Shigella: green without black
H2S production centre

Xylose-lysine deoxy cholate A differential media for Salmonella: red with black
(XLD) isolation of Shigella and centre
Salmonella from stool Shigella: red or clear

Campylobacter blood agar Selective media to isolate Appears pink grey moist when
(CAMPY-BA) campylobacter from stool incubated at 42℃

Selective Media Commonly Used to Recover diarrheal agents

CULTURE MEDIA PURPOSE PATHOGENS

Thiosulphate bile salt Vibrio species Yellow colonies; sucrose

sucrose agar (TCBS) Aeromonas species fermenting vibrio spp.
Such as V. cholera
Blue green colonies: non
sucrose fermenters as V.
vulnificus, parahaemolyticus

Cycloserine cefoxitin Selective media for Appears yellow from fructose

fructose agar (CCFA) Clostridium difficile fermentation

Sorbitol MacConkey agar A differential media to detect E.coli 0157:117 appear

(SMAC) sorbitol negative E.coli colorless
(contain sorbitol instead of
lactose)

● For routine culture media for Salmonella and Shigella the media used is Selenite-F Broth
or tetrathionate

6. How do you distinguish pathogenic from non-pathogenic bacteria in the stool culture?
• Normal flora (non-pathogenic bacteria) is already present in the body while pathogenic
microorganisms cause diseases.

7. What are the methods used to identify the pathogenic bacteria in the stool? Give at
least 2 methods.
● Routine culture for Enterohemorrhagic E.coli (E.Coli 0157:H7)
● Routine culture For Vibrio Spp.
● Routine culture media for Salmonella & Shigella

8. What method or procedures is used in performing blood culture?

● Closed system venipuncture; evacuated tube method (large amount of blood collected)

9. Is there an infection that goes to the blood that will definitely give a positive blood
culture? Give an example.
● Bacterial infection which spreads into the blood includes meningitis, osteomyelitis,
pneumonia, kidney infection, or sepsis. Septicemia is an infection that occurs when
bacteria enter the bloodstream and spread. It can lead to sepsis, the body's reaction to

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 the infection, which can cause organ damage and even death. Almost any type of germ
can cause septicemia. The ones most often responsible are bacteria, including:
Staphylococcus aureus., Streptococcus pneumoniae. E. coli.

10. How do you interpret and report blood culture? When is blood best collected?
● For blood culture, the incubation period is 7 days.
● Initial result should be released after 72 hours, interpreted and reported as “No growth
after 72 hours of incubation” if there is no growth of pathogens and “Growth of a particular
pathogen within 72 hours of incubation” if otherwise.
● Final result which will be released after 7 days will still be interpreted and reported as “No
growth after 7 days of incubation” if there is no growth of pathogens and “Growth of a
particular pathogen within 7 days of incubation” if otherwise.
● Blood samples are usually collected when the patient is in a febrile state - peak of his/her
fever.

CULTURE & SENSITIVITY

1. What is the method used in antibiotic sensitivity test? Name & describe other culture &
sensitivity test.
● DILUTION METHOD; various amount of antimicrobial substances incorporated into liquid
or solid media followed by inoculation of test bacteria
● DIFFUSION METHOD; place a filter disc/porous cup/bottomless cylinder measured
quantity of drugs on solid media that has been inoculated with heat bacteria

Other sensitivity test:

1. DISK DIFFUSION METHOD (KIRBY-BAUER TEST)
- In this test, the surface of a plate of special medium is spread with the test
bacterium producing a bacterial lawn, and small discs impregnated with a
pre-measured amount of antimicrobial are dispensed onto the bacterial
lawn.
2. BROTH DILUTION TEST
- This test challenges the bacterial isolate with antimicrobial agents in a liquid
environment. Various concentrations of an antimicrobial drug are
inoculated with a standard suspension of test bacteria. This test is used to
determine the MINIMUM INHIBITORY CONCENTRATION (MIC).
3. AGAR DILUTION METHOD / WELL DIFFUSION TECHNIQUE
- The organism and the microbial solutions are brought together on a culture
medium and this is used only in research studies because plate preparation
is laborious.
4. EPSILOMETER TEST (E-TEST)
- Utilizes a rectangular plastic strip device that contains a predefined
gradient of antibiotic concentrations that corresponds to MIC dilutions. This
plastic test device has a reading and interpretive scale indicated on the
surface of the strip. In this method, the strip is applied onto the surface of
an inoculated MHA plate. MIC is reported as the measure of the end of the
zone of inhibition

2. Describe the 2 different types of inoculum preparation for standardizing inoculum

suspension.
● Direct Colony Suspension
- Used for preparing inoculum from colonies grown within 18 to 24 hours or use for
bacteria that grow slowly or unpredictably

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 ● Growth Method
- Can be used by incubating the inoculated broth (with fast-growing bacteria)
within 2 to 6 hours.

3. Are all antibiotics used the same for all specimens? Explain.
• Antibiotics are not one-size-fits-all and the “broad-spectrum” antibiotics used to fight
infections in hospitals aren't the same as the very specific antibiotics the doctor may
prescribe to treat a bacterial ear infection. There are various antibiotics available and they
come in various different brand names. Antibiotics are usually grouped together based on
how they work. Each type of antibiotic only works against certain types of bacteria or
parasites. This is why different antibiotics are used to treat different types of infection.

4. What is the recommended culture media for antibiotic sensitivity testing? How thick
should the media be? How far should the antibiotics discs from one another and from the
edge of the petri plates?
● The recommended culture media for antimicrobial sensitivity testing is MHA (Mueller
Hinton Agar)
● Thickness of media: 3-4 mm
● Distance from each disk: ≥ 30mm
● Distance from the edge of the plate: ≥ 10mm

5. Explain the mechanisms of antimicrobial actions.

● Inhibition of cell wall synthesis - The antibiotics such as penicillins cephalosporins and
bacitracin and vancomycin inhibits the cell wall synthesis of the bacteria.
● Inhibition of protein synthesis - Chloramphenicol, erythromycin, tetracycline and
streptomycin target the bacterial ribosomes which are responsible for protein synthesis. If
protein synthesis is inhibited, its function in the bacterial structure will be compromised
leading to the death of the bacteria.
● Inhibition of Nucleic acid replication and transcription - Quinolones and Rifampin
inhibit DNA replication transcription. Particularly, quinolones inhibit the enzyme
topoisomerase which is required for DNA supercoiling.
● Injury to plasma membrane - Polymyxin B eliminates the semi-permeable characteristics
of the plasma membrane. As a result, the cell becomes devoid of nutrients leading to cell
death. Rifampin is used to treat pulmonary tuberculosis.
● Inhibition of synthesis of essential metabolites - Sulfanilamide and trimethoprim are
called anti-metabolites. They inhibit metabolic activity affecting enzyme activity. Bacterial
cells will not be able to produce essential metabolites in order for it to multiply.

6. Explain briefly antimicrobial resistance, sensitivity and slight sensitivity reactions?

● Sensitivity - the degree of drug effectiveness is characterized as susceptible also known
as sensitive means that the bacteria cannot grow if the drug is present or the antibiotic is
effective against the bacteria.
● Slightly sensitive or intermediate - means that a higher dose of antibiotic is needed in
order to prevent growth.
● Resistant - means the bacteria can grow even if the drug is present. It is also a sign of
ineffective antibiotics.

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BACTERIOLOGICAL ANALYSIS OF WATER
1. What are the 3 most important stages in water analysis?
● Presumptive phase, confirmed phase, completed phase

2. Describe different procedures in assessing water quality.

● The different procedures in assessing water quality are:
○ Multiple tube fermentation technique
- Role: detects & estimates the total coliforms in the water sample;
3 phases: presumptive phase, confirmed phase, and completed phase
○ Enzyme-substrate technique
- aka chromogenic substrate;
Role: for the detection of coliforms (TC) and E.coli (TTC)
○ Membrane filter technique
Role: this permits testing of large sample volumes, less preparation time
and allows isolation & enumeration of discrete colonies in bacteria
○ Heterotrophic plate count
- aka standard plate count or viable plate count;
Role: estimates the # of live bacteria and indicates the effectiveness of
water treatment

3. What is the normal colony count in water?

● Heterotrophic plate count levels in potable water should be <500 CFU/mL. These levels
may increase on occasion, but counts consistently >500 CFU/mL would indicate a general
decrease in water quality.
PARAMETER STANDARD VALUES

TOTAL COLIFORM Multiple Tube Fermentation Technique:

<1.1 MPN/100mL

Enzyme-Substrate Technique:
ABSENT or <1 MPN/100mL

Membrane Filter Technique:

< 1 total coliform colonies/100mL

THERMOTOLERANT Multiple Tube Fermentation Technique:

COLIFORM/E. COLI < 1.1 MPN/100mL

Enzyme-Substrate Technique:
ABSENT or <1 MPN/100mL

Membrane Filter Technique:

<1 thermotolerant coliform colonies/100mL

HETEROTROPHIC PLATE <500 est. CFU/mL

COUNT

4. What is the significance of high colony count in water?

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 ● The significance of high colony count in water suggests the presence of coliform bacteria,
specifically E. coli. This may also suggest the water may contain pathogens that can cause
an outbreak to the community e.g. diarrhea or any chemical and radioactive materials that
may cause lethal long term effects to people.

5. How do you identify the pollutant in water?

- Presence of coliforms is the principal indicator of the suitability of water for domestic,
industrial, or other uses. Experience has established the significance of coliform density
as a criterion of the degree of pollution and thus sanitary quality.

COLONY COUNT
1. What is the significance of high colony count in urine & water?
● Greater than 100,000 CFU/mL is highly indicative that the patient is suffering from urinary
tract infection.

2. What are the possible sources of error?

● The number of colonies obtained in a viable count depends not only on the inoculum size
but also on the suitability of the culture medium and the incubation conditions used; It also
depends on the length of incubation.
● The length of incubation.
● Some tiny colonies may be missed during the counting.
● The incubation conditions (medium, temperature, time)
● Key dilutions must be prepared.

3. Describe different methods of performing colony count.

• POUR PLATE METHOD: Simple to perform, colonies produced are relatively small &
compact,0.1 to 2.0 mL diluted samples are being used and reported as CFU/ML
• SPREAD PLATE METHOD: is limited by the small sample volume (0.1-0.5mL), uses
a spreader, needs to maintain a supply of suitable pre-dried agar and also reported as
CFU/ML
• MEMBRANE FILTER METHOD - this method permits testing large volumes of low-
turbidity water and is the method of choice for low count water. It has smaller display
area and can cause possible damage to cells due to excessive filtration procedure

FUNGAL SMEAR (KOH)

1. How do you prepare KOH smear from skin scrapings, sputum, urine, throat etc?
1. Place a small amount of material you wish to examine (e. g. skin scrapings, hair,
nail, pus and sputum) on a microscope slide.
2. Add 1 to 2 drops of 10% of KOH (preparation of wet mount).
NOTE: If material is hard, the slide is heated gently by passing over the flame.
Do this only after putting the coverslip.
3. Place a cover slip on the wet mount preparation and let it set for 10 to 30
minutes.
4. Examine under LPO and HPO respectively

2. Describe the chemical composition of KOH for fungal smear.

● The chemical composition of KOH includes:
○ 10 g anhydrous Potassium hydroxide

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 ○ 10 mL glycerol (to prevent KOH from crystallizing, not necessary in case, it is not
available)
○ 90 mL distilled water

3. Explain what makes KOH as an ideal mounting agent for fungal smear.
● KOH is preferred as a mounting agent on fungal smear since it destroys all non-fungal
cells or healthy cells leaving pathogenic fungal cells present in a smear. In addition, fungal
cells are resistant to digestion by KOH thus allowing it to be visualized under the
microscope.

4. What is the appearance of fungus in KOH smear?

● Fungus/dermatophyte can be identified in KOH smear by the presence of; fungal hyphae
(branched filaments) making up a mycelium, Arthrospores (broken-off spores),
Arthroconidia (specialised external spores), Spores inside a hair (endothrix) or outside a
hair (ectothrix). In addition, yeast cells have a budding appearance and a pseudohyphae
in which it has branched filaments similar to those of a dermatophyte forming a
pseudomycelium.

5. What is the significance of a positive KOH smear?

● Positive results generally indicate that a fungus is present and sometimes identify the type
causing an infection: Microscopic examinations (KOH prep or Calcofluor white stain): in
general, if fungal elements are seen, then a fungus is the likely cause of symptoms. These
tests, however, do not identify the fungus.

FUNGAL CULTURE
1. How do you prepare fungal smear from fungal culture?
● In preparation of fungal smear, KOH preparation is used to dissolve keratin in skin, hair,
or nail specimens.
○ Place a small amount of material to examine on a microscope slide.
○ Add 1 to 2 drops of 10% of KOH (preparation of wet mount).
○ If the material is hard, the slide is heated gently by passing over the flame. Do this
only after putting the coverslip.
○ Place a coverslip on the wet mount preparation and let it set for 10 to 30 minutes.
○ Examine under LPO and HPO respectively.

2. What are the compositions of lactophenol cotton blue dye?

● Lactophenol cotton blue (LCB) is composed of 20 ml lactic acid, 40 grams phenol crystals,
40 ml glycerin, 50 mg cotton blue dye and 20 ml distilled water.

3. What is the principle of hair baiting technique?

● Hair baiting technique uses keratin-rich hair particularly hair that hasn't been dyed or filled
with chemicals wherein it acts as a bait to know if there are presence of fungi
dermatophytes. These dermatophytes have keratinases that utilize keratin in order to
grow.
● Steps:
1. Add enough soil to a petri dish (2/3s or 3/4s to cover the bottom).
2. Using your finger (covered with gloves) make 3 or 4 depressions in the soil until
you can see the bottom of the petri dish.
3. Moisten the soil by adding sterile water.
4. With sterile forceps sprinkle some hair onto the soil, where some of the hair

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 should fall into the depression area.
5. Cover the petri dish and incubate at room temperature for 1 to 2 weeks.
6. Observe for fuzzy white mycelium growing on pieces of hair, record colonial
morphology.
7. Microscopically examine some of the hairs look for hyphae, and spores by
preparing an LPCB wet mount smears.

4. Why is the use of prepubertal hair necessary in hair baiting technique?

● Hair baiting technique, like in the name you are using keratin hair, preferably virgin hair
(hair that hasn’t been dyed or filled with chemicals because they are the type of hair rich
in keratin), and you’re going to use that hair sample as a bait to know and see if there are
presence of fungi dermatophytes because dermatophytes have keratinases that utilize
keratin

5. What is the principle of air exposure technique?

● The principle of air exposure technique determines if the environment you are working in
is sterile or not.
○ How will you know if it is sterile or not? Once there is no growth in your media
that was being exposed, it means that the environment is sterile, however if there
is growth, then definitely the atmospheric environment of wherever you’re working
is not that sterile.
● Steps:
1. Open a petri dish containing saboroud’s agar or potato dextrose agar in a desire
environment.
2. Leave the plates uncovered for 15 to 30 minutes.
3. Cover the plates and incubate at room temperature for 1 to 2 weeks.
4. Observe for filamentous colored mycelium growing on all areas of the plates
5. Record colonial morphology.
6. Microscopically examine the colonies look for hyphae, and spores by preparing
an LPCB wet mount smears.

6. What kinds of fungi were isolated in air exposure technique?

● When air exposure technique is done and airborne fungi like Cladosporium, Penicillium,
nonsporulating fungi, and Aspergillus were isolated; this only means that the
environment where you conduct the culture is unsterile.

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ADDITIONAL NOTES:
SPECIMEN MEDIA
Stool • MacConkey Agar
• Hektoen enteric Agar
• Xylose-lysine cholate Agar
• Campylobacter blood Agar
• Thiosulophate citrate bile salt sucrose Agar (TCBS)
• Cycloserine cefoxitin fructose Agar
• Sorbitol MacConkey Agar
Urine • BAP
• MacConkey Agar
• EMB
• MSA
• CNA (Columbia Nalidixic acid Agar)
• PEA (Phenylethyl alcohol)
• CLED (cysteine, lactose, electrolyte deficient)
CVUD • BAP
• MSA
• EMB
• CAP
• MacConkey Agar
• SDA* (Sabouraud Dextrose Agar)
Sputum • MacConkey Agar
• EMB
• BAP
• CAP
• SDA (Sabouraud Dextrose Agar)
• MSA

0.5 MacFarland standard:

• 0.05 mL 1% BaCl2
• 99.5 mL 1% H2SO4

Broad Spectrum - can kill or inhibit the growth of both Gram (+) and (-) bacteria
Narrow Spectrum - can kill or inhibit either Gram (+) & (-) bacteria
MIC - lowest concentration of a drug that inhibits growth of bacteria
MBC - lowest concentration of the drug that kills a microorganism

Urine Culture
Day 0 – specimen collection and streaking
Day 1 – culture, incubate, and perform biochemical testing
Day 2 – perform sensi, check and record microorganism growth
Day 3 – release the result

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 Multiple Tube Fermentation Technique
PHASE MEDIUM RESULTS
Presumptive phase Lauryl Tryptose Broth GET formation
(gas formation, effervescence,
turbidity)
Confirmed phase TC: Brilliant Green Lactose Bile Broth GET formation
TTC: EC Medium TC: 24-48 hrs at 35°C
TTC: 24 hrs at 44.5°C
Completed phase EMB, MAC, LTB GET formation

Enzyme-Substrate Technique
PRINCIPLE SUBSTRATE ENZYME RESULTS
Total Coliform Orthonitrophenyl-β-D- β-D- YELLOW color
Galactopyranoside (ONPG) Galactosidase
(coliform bacteria only)

Chromogenic substrate
Fecal Coliform 4-Methylumbelliferyl-β-D- β-Glucuronidase BLUE fluorescence
Glucuronide (MUG) (E. coli only)

Fluorogenic substrate

Compositions of lactophenol coton blue dye (LPCB)

Solutions Compositions Purpose
Lactic acid 20 ml Maintain pH
Prevent fungal multiplication
Phenol crystals 20 g Inhibit fungal multiplication
Glycerin 40 ml Prevent crystallization
Cotton blue dye 50 mg Stain
Distilled water 20 ml Solvent

Compositions of KOH for fungal smear

Solutions Compositions Purpose
10% Potassium 10 g anhydrous Potassium hydroxide
Hydroxide (KOH) 10 ml glycerol - Prevent crystallization
90 l distilled water

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