MICROBIOLOGY UNIT

PRE-TEST
MICROBIOLOGICAL INCUBATOR Is an insulated and enclosed device that provides an optimal
condition of temperature, humidity, and other environmental
conditions required for the growth of organisms. An incubator is a
piece of vital laboratory equipment necessary for the cultivation of
microorganisms under artificial conditions. An incubator can be
used for the cultivation of both unicellular and multicellular
organisms.
CO2 INCUBATOR A special kind of incubator that is provided with automatic control of
CO2 and humidity. This type of incubator is used for the growth of
the cultivation of different bacteria requiring 5-10% of CO2
concentration. Used for the growth of facultative/capnophilic
microorganisms. These are microorganisms that requires higher
amount of carbon dioxide for growth.
ELECTRIC INCINERATOR Uses infrared heat which aids for sterilizing inoculating loops and
needles.
BIOLOGICAL SAFEETY CABINET Is an enclosed, ventilated laboratory workspace to serve as a
means to protect the laboratory worker and the surrounding
environment from pathogens.
INOCULATING LOOPS AND NEEDLES Use for transferring and streaking of cultures.
CULTURE MEDIA Special media that contains nutritional and physical growth
parameters to promote microbial growth.
ANTIBIOTIC DISC A thin paper discs impregnated with an extract of antibiotics use for
testing susceptibility of a certain microorganism to antibiotic.
DENSITOMETER A machine use to measure the degree of cloudiness or
concentration of a particular solution to meet standard amount for
testing. Example is the inoculum for antimicrobial sensitivity testing
in Microbiology.
AUTOCLAVE It sterilizes laboratory equipment and media by heating them up to
a particular temperature for a specific period of time thus killing
bacteria, viruses and some spores by using steam under pressure.
The autoclave is considered a more effective method of
sterilization.
HOT PLATE WITH MAGNETIC STIRRER An electrically powered equipment for heating and agitation such us
during media preparation in laboratory. A magnetic bead is use for
agitation.

INTRODUCTION

Maraming bacteria ang nakakpagbigay sakit sa tao. Bagaman ang ilan ay maaring makita sa microscope, hindi
madaling alamin ang iba’t ubang klase nito. Upang malaman kug anong klaseng mikrobyo ang nakainfect sa
pasyente. Ang mga ito ay sinusuuri sa microbiology. Ang isang registered medical technologist ay ang mag iisolate
at magcuculture ng mikrobyo. Ito ay hahayaang tumubo sa isang culture media at lalagyan ng iba’t ibang uri ng
antibiotics. Upang malaman kung ano ang mabisa laban sa naturang mikrobyo. Ito ay makakatulong sa mga doctor
sa kanilang pagbigay lunas sa mga pasyente.
 SUMMARY O F PREANALYTICAL PHASE POLICIES AND GUIDELINES
MICROBIOLOGY TEST/ PROCEDURES
I. CULTURE AND SENSITIVITY TESTING
A. Aerobic Blood/Non-Blood
B. Fungal Culture (No Sensitivity Testing)
II. STAINING PROCEDURE
A. Gram Stain
B. Acid Fast Stain
C. India Ink Stain
III. OTHERS
A. KOH (Potassium Hydroxide) TEST (Slide Mount)
B. MTB Nucleic Acid and Rifampicin Resistance Detection (GENEXPERT)
CULTURE AND SENSITIVITY TESTING • All microbiology specimen must be placed in a CLEAN, DRY,
STERILE, WIDE MOUTH, LEAK-PROOF CONTAINER
• Collect aseptically BEFORE ANTIBIOTIC THERAPY.
• Complete the patient's demographics on request and indicate the
DIAGNOSIS
• All collected specimen must be submitted to the lab IMMEDIATELY.
• There is NO STAT request. Results vary from 3-7 days.
STAINING AND OTHER PROCEDURE • All microbiology specimen for staining must be placed in a CLEAN,
DRY, STERILE, WIDE-MOUTH, LEAK-PROOF CONTAINER
• Complete the patient's demographics on request and indicate the
DIAGNOSIS.
• For sputum AFB. "First morning" collection is ideal.
• For KOH Mount, infected site must be cleansed with 70% alcohol
before collection. Ointment or cream should not be applied.
• There is NO STAT request. Results are released within the day/ shift.
MTB NUCLEIC ACID AND RIFAMPICIN • All collected specimen must be submitted to the lab IMMEDIATELY.
RESISTANCE DETECTION • For patients whom there is clinical suspicion of tuberculosis (TB).
(GENEXPERT) • Specimen and volume requirement is 2 ml sputum or endotracheal
aspirate.
• Alternative specimen is NOT VALIDATED.
• Rejection criteria are QNS, improperly labeled specimen, specimen
stored and transported outside the required temperature, blood
streaked/ bloody specimen.
• Running day is every Mondays and Thursdays (Batch Running).
• Cut-off time is 10:00 AM.
• TAT/ Releasing of results is same day if received within the unning
day's cut-off time.

GRAM STAINING
1. Label a clean slide with the specimen log number.
2. Use a sterile loop or autoclave applicator stick to get a sample from the specimen container
3. Smear the sample on the slide and let it dry.
4. Heat fix the slide 2-3x over a flame
5. for stainig procedure, follow the product insert.
6. Put a few drops of crystal violet in the smear an let it stand for 5 mins.
7. After 5 mins wash with water
8. Put a few drops of gram’s iodine and let it stand for 2 mins. Then, wash with water.
9. Put a few drops of gram’s decolorizer for 30 seconds. Then, wash with water.
10. Lastly, Put a few drops of safranin and let it stand for 30 seconds.
11. Tap the excess water and dry. Observe the slide under the microscope.

ACID FAST STAINING (ZIEHL-NEELSEN METHOD/ HOT METHOD)

1. Label a clean slide with the specimen log number.
2. Smear the sample on the slide using sterile loops or applicator stick making a 3 cm by 2 cm size of smear
3. Coil the smear using a pointed tip of a barbeque stick starting form the ceneter of the smear outward.
4. Let it dry for a few minutes. Before staining, heta fixed the smear 2-3x over a flame.
5. for stainig procedure, follow the product insert.
6. Cover the smear with carbol fuchsin then steam the slide with flame until fumes come out over the stain and
let it stand for 10 mins.
7. After 10 mins, rinse off the stain with water.
8. Decolorize the smear with acid alcohol for 2 mins or until the smear becomes colorless then rinse with
water.
9. Lastly, cover the smear with methylene blue and let it stand for 5 mins
10. After 5 mins. Rinse with water. Tap the excess water and let it ry. Observe under the microscope.
 SPECIMEN PROCESSING

Simple or Parallel Uninterrupted Streaking method

 ACTIVITY 1
A specimen for Microbiology analysis was not sufficient
for the desired request. What will you do.
a) Proceed and report with invalid result.
b) Inform the requesting ward that the specimen
was not sufficient and must send with sufficient
specimen.
c) Discard the specimen immediately then inform
the requesting ward.
d) Proceed even the specimen was not sufficient.
A doctor insisting a blood culture request for a patient
who already taken an antibiotic 2 days prior to the test.
The laboratory informed the doctor that there is no
available blood culture bottle with Antibiotic Removal
Device (ARD). What will you do?
a) Refer the situation to the pathologist on duty.
b) Proceed with the request.
c) Remarks that the patient has taken antibiotics
and proceed.
d) Ignore the doctor’s request.
A wound swab culture and gram stain is requested but
the specimen submitted was 1 swab only. What will you
do?
a) Inform the requesting ward to prioritize culture.
b) Ignore the tests.
c) Cancel the request
d) Proceed with the tests requested.
A blood culture was requested with on-going previous
blood culture request. What will you do?
a) Discard the current request.
b) Proceed without informing the requesting ward.
c) Cancel without informing the requesting ward.
d) Call the requesting ward that it has a previous
on-going request and ask if the current request
will continue.
Inform the requesting ward that the specimen was
not sufficient and must send with sufficient
specimen.
Insufficient specimen will give erroneous results if it
does not meet the required volume of a test.
Refer the situation to the pathologist on duty.
During situation that cannot be resolve between the
requesting physician and the medical lab scientist, it is
advisable to refer to your pathologist on duty to further
explain the effect of antibiotic treated patients to its
culture result.
Inform the requesting ward to prioritize culture.
2 separate swabs is needed to perform different test.
Culture will best identify whether the patient’s illness
was cause by certain bacteria
Call the requesting ward that it has a previous on-
going request and ask if the current request will
continue.
The requesting ward must be informed to confirm if they
need another set of request with same test and to avoid
confusion during reporting of results.
A pleural fluid culture specimen with culture request and Process the specimen first to Microbiology for
cytology (for histopathology) request was brought in the culture then forward the specimen to
laboratory. But only 1 specimen was given. What will Histopathology for cytology test.
you do?
a) Give the specimen to Histopathology then to All shared specimen must be given first to Microbiology
Microbiology after the cytology test. to avoid contamination of specimen for culture
b) Put a fixing agent immediately on the specimen
for cytology.
c) Ignore the culture request.
d) Process the specimen first to Microbiology for
culture then forward the specimen to
Histopathology for cytology test.

ACTIVITY 1
Culture Media: Triple Sugar Iron Agar 16.25
Distilled Water: 250mL

Culture Media: Alkaline Peptone Water 4

Distilled Water: 200mL

Culture Media: Tryptic Soy Broth 6

Distilled Water: 200mL
Culture Media: Brain Heart Infusion Broth (BHI) 18.5
Distilled Water: 500mL

Culture Media: Lysine Iron Agar (LIA) 10.38

Distilled Water: 300mL

Culture Media: Mueller Hinton Agar 28

Distilled Water: 1000mL
Culture Media: Mac Conkey Agar 24.77
Distilled Water: 500mL

Culture Media: Simmons Citrate Agar (CIT) 3.64

Distilled Water: 150mL
Culture Media: Fluid Thioglycollate Medium 2.98
Distilled Water: 100mL

Culture Media: Bile Esculin Agar (BEA) 6.35

Distilled Water: 100mL
 SHIFTING EXAM
Which of the following shows a positive urease test?
a) Klebsiella
b) Proteus
c) None of these
d) Both A and B
The bacteria involved in the production of dental caries?
a) Both A and B
b) S. sanguis
c) S. pyogenes
d) S. mutans
A pure culture refers to: A culture which contains only a single type of
a) A culture containing bacteria that all have the organism.
same shape.
b) A culture which causes only a single disease.
c) A culture which has never been used to
inoculate a patient.
d) A culture which contains only a single type of
organism.
Pseudomonas aeruginosa forms a blue water soluble Pyocyanin
pigment called
a) B-carotene
b) Cholororaphin
c) Pyocyanin
d) Pyoverdin
Which gram negative bacilli causes dysentery? Shigella species
a) Pseudomonas aeruginosa
b) Shigella species
c) Klebsiella species
d) Proteus species
Which of the following is/are oxidase-positive bacteria? All of these
a) Vibrio
b) Neiserria
c) All of these
d) Pseudomonas
What type of antibiotics work by affecting cell wall Beta-Lactam Antibiotics
synthesis?
a) Tetracyclines
b) Beta-Lactam Antibiotics
 c) Macrolides
d) Aminoglycosides
Ringworm is caused by which fungi? Dermatophytes
a) Borellia burgdorferi
b) Dermatophytes
c) Aspergillus species
d) Candida albicans
Which of the following inferences indicates the ability of Both A and B
an organism to utilize citrate as a sole source of carbon
in Simmon’s citrate medium?
a) None of these
b) Blue color
c) Appearance of growth
d) Both A and B
A genus of bacteria that causes diseases in humans. Mycobacterium
a) Mycobacterium
b) Bacillus
c) Lactobacillus
d) Bifidobacterium
Gram-positive bacteria responsible for food poisoning Clostridia
a) Mycoplasmas
b) All of these
c) Clostridia
d) Pseudomonas
A series of sequential dilutions that reduce a dence Serial dilution
culture of cells into a more usable concentration is called
a _________.
a) Serial dilution
b) Sequential dilution
c) Water electrolysis
d) Biodegradation
The scientist who first discovered the substance Alexander Fleming
penicillin was
a) Joseph Lister
b) None of these
c) Paul Ehrlich
d) Alexander Fleming
Which of the following fields is concerned with the study Mycology
of fungi?
a) Physiology
b) Mycology
c) Bacteriology
d) Virology
The coagulase test is used to differentiate Staphylococcus aureus from Staphylococcus
a) Streptococcus pyogenes from Enterococcus epidermidis
faecalis
b) Streptococcus pyogenes from Staphylococcus
aureus
c) Staphylococcus aureus from Staphylococcus
epidermidis
d) Staphylococcus epidermidis from Neisseria
meningitidis
Which of the following shows positive result for All of these
phenylalanine deaminase test?
a) Morganella
b) All of these
c) Providencia
 d) Proteus
Staining procedure designed for bacterial families with Acid fast staining
high lipid content and resistant to standard staining is?
a) Gram stain
b) Acid fast staining
c) Negative staining
d) Simple stain
It is the study of both causes and effects of diseases on Pathology
the body.
a) Etiology
b) Morphology
c) Pathogenesis
d) Pathology
Which of these is NOT a gram negative bacteria? Streptococcus pneumoniae

a) Streptococcus pneumoniae
b) H. influenzae
c) E. coli
d) Salmonella
Mac-Conkey medium is an example of Differential medium
a) All of these
b) Enrichment medium
c) Transport medium
d) Differential medium
What is the difference between an antibody and an An antigen activates an antibody; an antibody is
antigen? produced by the immune system.
a) An antigen activates an antibody; an antibody is
produced by the immune system.
b) Antigens can be measured by ELISA.
c) Antibodies can be measured by IGRA.
d) An antigen is produced by the immune system;
an antibody activates the antigen.
Viruses require _______for growth Living cells
a) Bacteria
b) Plants
c) Animals
d) Living cells
A student is asked to sub-culture a bacterial organism Obligate aerobe
onto fresh media for future studies. However during sub-
culturing the student locks the test tube preventing
oxygen flow into the media. After days, the culture died.
What type of culture did the student have?
a) Strict anaerobe
b) Facultative anaerobe
c) Obligate aerobe
d) None of the above
Which of these gram negative bacteria commonly cause Escherichia coli
a UTI?
a) Neisseria meningitidis
b) Escherichia coli
c) Neiserria gonorrhea
d) Salmonella
Which of the following gram-positive bacteria causes Streptococcus
pharyngitis (sore throat)?
a) Streptococcus
b) Mycobacterium
c) Neiserria
 d) Staphylococcus
The primary stai of Gram’s staining method is Crystal violet
a) Safranin
b) Crystal violet
c) Methyl red
d) Phenolpthalene
Microorganisms that can only reproduce by using a host Viruses
cell.
a) Sphirochetes
b) Mycobacteria
c) Bacteria
d) Viruses
Clostridia species eg. C. tetani, C. perfringens, C. Tetanus, gas gangrene, botulism
botulinum are anaerobic gram positive bacilli that
causes:
a) Tetanus, gas gangrene, botulism
b) Endocarditis
c) Diptheria
d) Tonsilitis, cellulitis, scarlet fever, septicemia
Which of the following disinfectant is effective against
viruses?
a) Formaldehyde
b) Hypochlorite
c) Hydrogen peroxide
d) All of these
Which of the following is NOT a characteristic of yeasts?
a) Yeasts can grow in absence of oxygen.
b) Yeasts make food from sugar.
c) Yeasts are unicellular fungi.
d) Yeasts grow best in moist environments of 25-
30 degrees Farenheit.
Which of the following bacteria can grow in acidic pH? Lactobacilli
a) Shigella
b) Vibrio cholerae
c) Salmonella
d) Lactobacilli
Which is NOT true of bacteria?
a) They are prokaryotes
b) They have nucleus
c) Some use photosynthesis
d) They have peptidoglycan in their cell walls
Acute diarrhoeal disease resembling cholera can be
caused due to
a) V. alginolyticus
b) V. vulnificus
c) All of these
d) Aeromonas hydrophila
Which of these is commonly found on the skin? Staphylococcus aureus
a) Streptococcus pneumoniae
b) Staphylococcus aureus
c) Clostridia species
d) Viridans streptococci
Which of the following is a primary stain for acid fast Carbol fuschin
staining of mycobacteria?
a) Carbol fuschin
b) Methylene blue
c) Crystal violet
 d) Geimsa
Catalase production is negative in which of the Streptococcus
following?
a) Salmonella
b) Proteus
c) Staphylococcus
d) Streptococcus
Temperature below that of optimal growth cause Slow their growth rate as temperatures approach
bacterial cultures to: the minimum
a) None of the above
b) Thrive without any evident damage to the cell
c) Perish quickly at high temperatures
d) Slow their growth rate as temperatures
approach the minimum
What type of fluid is added to the autoclave? Distilled water
a) Tap water
b) Normal saline
c) Sterile water
d) Distilled water
A Mueller-Hinton plate is mainly for Susceptibility testing
a) Organism identification
b) Susceptibility testing
c) Fungal elimination
d) Yeast culture
Agar is used for solidifying culture media because All of the above
a) It does not affect by the growth of bacteria
b) All of the above
c) The melting and solidifying points of agar
solution are not the same
d) It does not add to the nutritive properties of the
medium
________ are used in laboratories to sterilize glassware, Autoclaves
media and even waste.
a) Centrifuges
b) Incubators
c) Autoclaves
d) Biosafety cabinets
Each of the following agents is a recognized cause of
the diarrhea except
a) Enterococcus faecalis
b) Clostridium perfringens
c) Escherichia coli
d) Vibrio cholerae
Cauliflower appearance on Lowenstein-Jensen medium Mycobacterium tuberculosis
is produced by
a) None of these
b) Both A and B
c) Mycobacterium bovis
d) Mycobacterium tuberculosis
Streptococcus pyogenes can be differentiated from other Bacitracin sensitivity
haemolytic Streptococci on the basis of
a) Bacitracin sensitivity
b) Penicillin sensitivity
c) Aminoglycosides sensitivity
d) Erythromycin sensitivity
Which of the following is/are not a gram-positive Pseudomonas
bacteria?
a) Streptococcus
 b) Mycobacteria
c) None of the above
d) Pseudomonas
A disease producing species occurring in a mixed Pathogenic properties
culture can often be selected on the basis of its
a) Special nitrogen source
b) None of the above
c) Pathogenic properties
d) Special carbon source
The most toxic exotoxin is Botulinum toxin
a) Tetanus toxin
b) Botulinum toxin
c) Diphtheria toxin
d) Cholera toxin
Typical drumstick appearance of bacilli is generally seen C. tetani
in
a) C. botulinum
b) C. tetani
c) C. perfringens
d) C. histolyticum
Which of the following medium are used to differentiate Thiosulfate-citrate-bile-sucrose
the colonies of Vibrio cholerae and V.
parahaemolyticus?
a) MacConkey
b) All of the above
c) Thiosulfate-citrate-bile-sucrose
d) Alkaline bile salt
_________ contracted by inhaling spores are not
actually contagious from person to person or animal to
person.

a) Fungi
b) Bacteria
c) Viruses
d) Parasites