METHODS OF STUDYING
consistency which prevent oxidation and drying
BACTERIA
General Specimen Guidelines
1. Collect the specimen in the acute phase of infection
& before antibiotics is administered.
2. Specimen should be collected from the actual site of
infection.
3. Specimen should be collected using proper technique
and supplies minimal contamination from the normal
flora.
 For Aerobes - swab, needle aspiration.
 For Anaerobes - needle aspiration and tissue.
4. Collect the appropriate quantity of specimens.
5. Package the specimen in a container or transport
medium designed to maintain the viability of the
organism & avoid hazards the result from leakage.
6. Label specimen accurately
7. Transport the specimen to the lab promptly or make
provisions to store the specimen- will not degrade the
suspected organism.
8. Notify the lab in advance if unusual pathogens or
agents of bioterrorism are suspected.
• Anthrax - Used as a bioterrorism agent on
9/11.
Transport Media
Transport Media
It has the nutrients to maintain the growth of the
organism, a buffer to maintain pH, and a small amount of
gas to maintain moisture.
1. Carry Blair
2. Stuart’s
3. Amies
4. Transgrow and JEMBEC
5. Todd Hewitt
6. LIM: modified Todd-Hewit
7. Buffered Glycerol Saline
Transport Media - To maintain
viability
1. Carry Blair - for stool
• Remel Cary Blair Transport Medium is a
semisolid medium recommended for use in the
transportation and preservation of clinical
specimens, primarily stool and rectal swabs.
• This medium has a low nutrient content to
prevent replication of organisms while
maintaining viability
2. Stuart’s
• Stuart Transport Medium is a non-nutritional
medium which maintains the viability of
organisms without significant multiplication.
• The small agar content provides a semisolid
during transport.
3. Amies
• Amies is an improved transport medium
containing charcoal to prolong the viability of
pathogenic organisms.
o Charcoal - absorbs fatty acids.
• It is a semi-solid medium recommended for
qualitative procedures for transporting clinical
swab specimens to the laboratory.
4. Transgrow or JEMBEC
• The JEMBEC system is made up of a rectangular
plastic tray which contains modified Thayer-
Martin medium.
o Thayer-Martin Medium - use CAP
o Chocolate Agar Plate (CAP)
• The tray has a well into which a CO2-generating
tablet is placed.
• The tray is placed inside a zip lock plastic bag..
5. Todd Hewitt
• Todd Hewitt Broth is used for the cultivation of
streptococci and other fastidious microorganisms.
• This medium is very nutritive by the presence of
peptic digest of animal tissue and beef heart
infusion. Dextrose stimulated hemolysin
production.
• This medium is well buffered by sodium
phosphate and sodium carbonate to neutralize the
acid produced during dextrose fermentation.
6. LIM broth/ Modified Todd-Hewitt
• It is also known as the Todd Hewitt Broth w/
Colistin and Nalidixic Acid.
Colistin
o Used for Gram Negative Bacilli
o No effect on Gram Positive
Nalidixic Acid
o Effective for Gram Negative
o Has minor Gram Positive activity.
• It is a selective enrichment medium which may
be used in qualitative procedures to isolate Group
B Streptococci from clinical specimens
containing mixed bacterial flora.
Group B Streptococci
o Streptococcus agalactiae - gram
positive
7. Buffered Glycerol Saline
• Buffered Glycerol Saline is used for collection
and transportation of fecal specimens.
o The prepared medium for Buffered
Glycerol Saline has:
o Light pink color - indicates Alkaline
pH.
o Yellow - acidic; should be discarded.
Because of unfavorable effect on gram
negative Bacilli especially for Salmonella
spp.
1
• The medium contains:
1. Sodium Chloride - acts as nutrients;
provides essential ions.
2. Dipotassium and Monopotassium
phosphate - acts as buffers
 3. Phosphate buffers - used with Glycerol to
recover pathogenic bacteria.
Specimen Collection Equipment
• Dacron and Rayon - good for bacteria and
viruses.
Swab recommended for:
o Virus: Dacron
o Urogenital Specimen: cotton swab with
charcoal
o Swab not for fungi and anaerobes.
• Swab Aerobic
o Cotton Swab - toxic to Neisseria, good for
virus.
o Calcium alginate swab - toxic to virus,
good for Neisseria.
Transport Temperature
• Cerebrospinal Fluid (CSF) - transport temp -
room temp (RT), storage temp - body temp (35-
37°C)
• Urine, stool, swab, viral specimens, sputum,
foreign devices: 4° - Ref temp (0 - 4°C)
• Serum for serology: -20°C for 1 week
• Tissue specimen for long term storage: -70°C
• Specimen suspected to contain ANAEROBE
SHOULD NOT BE REFRIGERATED.
o Refrigerated temp - inhibits the viability of
certain anerobic microorganisms.
• Refrigerated Temperature
o Stool, sputum, urine and swab except
genital.
• Room Temperature
o Cerebrospinal Fluid (CSF), blood, body
fluids, genital swab of Neisseria
gonorrhea.
Anticoagulants/Preservatives
• To maintain accurate colony count, if there is a
delay in processing urine, this preservative may
be added to SPS.
• 0.025% Sodium polyanethol sulfonate (SPS)
- to prevent clotting, anti-complementary, anti-
phagocytic, neutralizes aminoglycoside.
• HEPARIN - anticoagulant used in VIRAL
CULTURE, may inhibit gram (+) and yeast.
• EDTA - VIRAL Polymerase Chain Reaction
(PCR) anticoagulant -- for SARS - COV - 2
• Not to be used in microbiology laboratory:
EDTA and Citrate.
ANEROBIC JAR
Components of GasPak Anerobic System
1. Polycarbonate jar (or anerobic container)
2. A lid with a gasket to prevent airflow
3. Indicator strip
4. Disposable gas-generating pouch
5. A palladium catalyst
Indicator of Anaerobiosis
The effectiveness of GasPak anerobic system can be
checked by the following methods:
Anerobic indicator strips:
1. Impregnated with methylene blue: remains
colorless in anaerobic conditions, but turns
blue on exposure to oxygen.
2. Impregnated with resazurin, a redox
indicator: colorless (white) in anerobic
conditions but turns to pink in the presence of
oxygen.
Swab
Specimen for Microbial Analyses
Specimen Collection Requirement for Specific Sites:
A. Blood Culture
• Antiseptic: Alcohol - Iodine - Alcohol
• Anticoagulant: 0.025% SPS
o Neutralizes bactericidal effect of human
serum.
o Prevents phagocytosis
o Antibiotic removal capability
o 2 sites are usually requested
• Children = 1:5 to 1:10
• Adult = 1:10 to 1:20
0.025% SPS inhibits certain bacteria such as:
• Neisseria spp.
• Gardnerella vaginalis - causative agent of
bacterial vaginosis.
• Pepto streptococcus anaerobius
• Staphylococcus moniloformis
0.025% SPS + 1% gelatin to not inhibit the bacteria
mentioned.
• Subculture on Blood Agar Plate (BAP),
Chocolate Agar Plate (CAP) and, MacConkey
Agar (MAC)
- Turn Around Time (TAT) - 7 days = time of
release of the specimen.
 21 days - Brucellosis, Endocarditis,
Subacute Bacterial Endocarditis (SBE)
 8 weeks - Leptospirosis
• Automation: Uses BACTEC 9120
 TAT: 5 days
 Medium: BACTEC Broth (1:10 ratio)
with SPS.
Note: 0.025% SPS (liquid) - anticoagulant, anti-
complementary, anti-phagocytic, neutralizes
aminoglycosides and bactericidal effect on serum.
• Bacteremia: small amount of bacteria in the
blood.
o Transient - Normal flora introduced into the
blood.
o Intermittent - Bacteria sporadically
discharged from extravascular abscesses or
infections into the blood
o Continuous - Constant release of bacteria into
the blood.
• Septicemia: large number of bacteria;
multiplication of bacteria in the blood.
• Also known as Blood Poisoning Disease
B. Throat and Nasopharyngeal Cultures
Culture must include anerobic conditions for beta
Streptococcus; Group A and B Streptococcus: beta
Streptococcus.
- Group A: Streptococcus pyogenes
- Group B: Streptococcus agalactiae 2
 UPPER PART:
1. Throat
o Group A (Streptococcus pyogenes)
Streptococcal Infection; causes strep. throat
 o Oral Thrush - Candida albicans; Fungi
o Epiglottitis - Haemophilus influenzae type B
o Oral Gonorrhea - Neisseria gonorrhoeae
 Most abundant Throat Flora:
Viridans Strep or α-Hemolytic Streptococcus
 Most Common Throat Pathogen:
Streptococcus Pyogenes or β-hemolytic Strep
or Group A Streptococcus
2. Nasopharynx
• Nasal Swab
- Moistened swab in the nares/nose; detects
carrier state of Staphylococcus aureus
• Nasopharyngeal Swab
- Flexible swab inserted through the nose
and into posterior nasopharynx and then
rotated for 5 seconds.
- Detect carrier state of Neisseria
meningitidis and for detection of
Bordetella pertussis.
 LOWER PART:
1. Sputum
o Bronchitis or Pneumonia
o Collected by a deep cough
o Specimen with too many epithelial cells
and/or few PMN are not suitable for culture.
CLINCAL SPECIMEN FOR THROAT AND
NASOPHARYGEAL
- May often be contaminated with normal flora so it
is important to evaluate the quality of the specimen
- Ideally collected in the morning; rinse mouth or
gargle with water then instruct the patient to deep
cough and expectorate into a sterile container.
- Three morning specimen sputum specimens for
Acid Fast Smears; Morning specimen are
preferred since they are more concentrated
- Should be Gram stained and checked for acceptability
- check for epithelial cell and polymorphonuclear cells
Barlett’s Classification - assess the quality and for
scoring of sputum
- In Barlett’s classification, the number of
neutrophils and epithelial cells per low-power field
(LPF) is enumerated. Based on these findings, the
sputum is given a score. Scores of 0 or less indicate
a lack of inflammation or the presence of saliva
while scores greater than 1 indicate inflammation
or infection.
Number of Neutrophil per LPF 28 +2
Number of EC per LPF 26 -2
Total Score 0
Interpretation: Lack of inflammation or presence of saliva.
Number of Neutrophil per LPF 1 0
Number of EC per LPF 9 0
Total Score 0
Interpretation: Lack of inflammation or presence of saliva.
Number of Neutrophil per LPF 28 +2
Number of EC per LPF 6 0
Total Score +2
Interpretation: Have inflammation
AMs - Alveolar Macrophages
CCs - Columnar Cells
C. Urine Cultures
• UTI (Cystitis, urethritis) - lower UTI
• Cystitis - Infection of Urinary Bladder
• Urethritis - infection of Urethra
• Pyelonephritis, glomerulonephritis) - upper UTI
• Kidneys - severe
Type of Urine Specimen:
1. Midstream Clean-catch urine
2. Catheterized Urine
3. Suprapubic Aspiration
Urine (Random, Catheterized, Midstream,
Suprapubic)
- Specimen of choice for bacterial culture Midstream
Clean-catch.
- Catheterized for those unable to produce.
o Foley catheter - Urine
- Ideally, first morning urine is used since it is more
concentrated.
- Must be preserved or refrigerated if not processed,
suitable preservative BORIC ACID to maintain
accurate colony count.
- Suprapubic Aspiration - Collected via needle
aspiration above the symphysis pubis; anerobic
process.
 Major cause of UTI: Escherichia Coli
 Cause of UTI among sexually active young
Females: Staphylococcus saprophyticus
CLINICAL SPECIMEN FOR URINE
- MAC and BAP are suitable combination.
- Urine specimen must be culture within one hour or
refrigerate - however, never refrigerate for longer than
24 hours
- Colony count should be performed on all urine sample;
when performing colony count use CALIBRATED
LOOP (1µL or 10µL)
- Formula to compute for colony count per mL of urine:
 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 × 𝐷𝐹 × 1000 (If
1µL loop was used) = colony count/mL of
urine.
 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 × 𝐷𝐹 × 100 (If
10 µL loop was used) = colony count/mL of urine
 Considered significant: Greater than or equal
to 100,000 colony count/mL or 𝟏𝟎𝟓
Example:
If a 1µL calibrated loop yields 145 colonies. What is
colony count/mL of urine?
145 × 1000 = 145, 000 𝑜𝑟 1.45 × 105 CFU/mL urine
3
If a 10µL calibrated loop yields 1450 colonies. What is
colony count/mL of urine?
1450 × 100 = 145, 000 𝑜𝑟 1.45 × 105 CFU/mL urine

D. Gastrointestinal Tract
• Stool Specimens.
- Do not gram stain; for detection of
Enteric Pathogen
- 1 - 2 grams stool on sterile vial
- May be directly plated on differential plate
(Eosin-Methylene Blue agar) EMB or MAC
and onto a selective media (Hektone Enteric
Agar) HEA and (Salmonella Shigella Agar)
SSA and enrichment like tetrathionate.
- Routinely screen for Salmonella, Shigella,
Campylobacter.

• Rectal Swabs
- Rectal swab on Cary Blair
- Collected by inserting a swab 2.5cm past
anal sphincter. Feces should be visible on the
swab.

E. Cerebrospinal Fluid (CSF)

- 3rd or 4th puncture in between the 3 lumbar
vertebra.
- Clear and colorless
- Examine immediately or hold at 37 °C for
no longer than 6 hours
- Centrifuge, use sediment for:
 Smears, G/S, India ink Culture:
Haemophilus influenzae, Neisseria
meningitidis.

CLINICAL SPECIMEN FOR CERBROSPINAL

FLUID
- Usually collected using 3-4 tubes
- Tube #2 is for Microbiology, gram stain, and
culture, if with the 4th tube, use them for better
exclusion of skin contaminants.

1st tube for Chemistry

2nd tube for Microbiology
3rd tube for Hematology
4th tube for Serology or Microbiology

- Examine immediately, Priority/1st to process or

hold either at 37 °C storage temp; transport temp:
room temperature.
- Routine test: India ink and gram stain
- Isolation of Neisseria meningitidis, Streptococcus
pneumoniae and Haemophilus influenzae.
- Centrifuge: sediment for culture and GS.

F. Genital Tract
STI’s include infection caused by: Neisseria
gonorrheae, HSV type 2, Chlaydia tracomatis,
Trichomonas vaginalis, Gardnerella vaginalis,
Candida albicans.

Specimen:
• Male: urethral discharge
• Female: cervical or urethral discharge
• Use of selective media: Martin-Lewis or
modified Thayer Martin. 4
G. Wounds and Abscesses
Use of swab, syringe, and needle 2 types of wound
infections:
o Exogenous - result from animal & human bites,
burns, ulcers, traumatic wounds.
o Endogenous - attributed to indigenous bacteria.

H. TB Culture - 3 morning specimen

- 1 sputum for GS; 2 sputa for AFS (ACID FAST
STAIN)
- Gold Standard: NALC and NaOH (Sodium
Hydroxide)
- NALC (n-Acetyl-L-Cystine): Digestant/mucolytic
- 2.4% NaOH: decontamination
- Oxalic acid - Pseudomonas contamination (cystic
fibrosis)

Criteria for Specimen Rejection

 Any specimen received in formalin.
 A single swab with multiple requests.
 Submission in an improper, nonsterile container in
which portions of the specimen have leaked out.
 Specimens that are obviously contaminated as
evidenced by the presence of foreign materials.
 Improper transport medium.
 Information the requisition slip not matched with the
specimen label.
 No patient ID on specimen container.
 Quantity of specimen is inadequate to perform all
tests requested.
 Specimen transport more than 2 hours & specimen
was not preserved.

Critical Values
 Positive blood cultures, CSF Gram Stain or culture
and positive acid-fast stain.
 Streptococcus pyogenes in surgical wound
 Gram stain suggestive of gas gangrene.
 Blood smear for patient with malaria.
 Positive cryptococcal antigen test
 Detection of select agents or other significant
pathogens (antibiotic-resistant strain)