MEDT 25 – CLINICAL INTERNSHIP 1 (EACMC) 1

6th ROTATION: MICROBIOLOGY (MS. GRACIA M. CATULIN, RMT)

MICROBIOLOGY 7. Measure the blood in a sterile test tube or use a sterile serological pipette or a
CULTURE MEDIA PREPARATION graduated cylinder.
TYPES OF CULTURE MEDIA 8. Mix by gentle swirling to avoid formation of bubbles.
A. ACCORDING TO CONSISTENCY 9. Dispense/ pour about 20 mL into sterile petri dishes. Depth of agar layer must
1. Liquid (Broth) Media be 4mm.
o Propagation of large number of organisms, fermentation studies, and 10. Once solidified, label the plates with the name of the media and preparation date.
various other tests. 11. Store the plates at 4 deg. C in an inverted position or for longer shelf life, place
o E.g., BHI broth, thioglycolate in a sterile tight plastic bag (for up to 2-3 months).
2. Semi-Solid Media
o Cultivation of microaerophilic bacteria, or for determination of bacterial QUALITY ASSURANCE
motility. I. STERILITY TEST
o E.g., SIM, LIM 1. Incubate two tubes or plates from each autoclaved or filter-sterilized medium
3. Solid Media overnight at 36 ± 1 deg. C.
o Allows media to grow in physically informative or useful ways. 2. Check tube or plate for growth after 24 hours of incubation.
o E.g., MHA, NA, BAP
II. CULTURE RESPONSE TEST/ GROWTH DEPENDENCE TEST
B. ACCORDING TO FUNCTION ▪ For plated media:
1. General (Basal/ Basic) Media o Inoculate at least one strain to test for ability of a media to support growth of
o Supports the growth of a wide variety of microorganism types and lack the target pathogen.
inhibitory property. ▪ For biochemical media:
o E.g., Na, TSA o Inoculate at least one organism that will produce a positive reaction and at
2. Selective Media least one organism that will produce a negative reaction.
o Favors the recovery of specific types or genera of microorganisms and ▪ Use quality assurance organisms such as Staphylococcus aureus ATCC
inhibits other members of a mixed microflora. 25923, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC
o E.g., MAC, TCBS, GBA, BCA 27853, or organism isolate from clinical specimens that were previously well-
3. Differential/ Indicator Media identified.
o Possess certain ingredients that enable presumptive identification of a ▪ Record quality control results as to whether the strain used produces the
specific genus or species either from a pure or mixed culture. appropriate biochemical reactions/ color on the test medium.
4. Enriched Media
o Supports the growth of a wide variety of microorganisms including some PROPER COLLECTION, TRANSPORT, AND PROCESSING OF SPECIMENS
of the fastidious ones. How to successfully recover bacteria from clinical specimens?
o E.g., BAP, CAP ▪ Advance planning
5. Enrichment Media ▪ Collect appropriate and adequate amount of specimen.
o Used to increase the relative concentration of certain microorganisms, ▪ Proper packaging and transport.
including some of the more fastidious ones. ▪ Ability of the laboratory to accurately perform the diagnostic test.
o E.g., thioglycolate broth, selenite F broth
6. Transport Media General Guidelines:
o For temporary storage of specimens being transported to the laboratory 1. Follow universal precaution guideline.
for cultivation. 2. Treat all specimens as potential biohazards.
o E.g., Cary Blair Transport Medium, Amies Transport Medium 3. Collect specimen before antibiotic treatment
4. Collect from appropriate sites.
SAFETY PRECAUTIONS 5. Practice proper and aseptic collection technique.
▪ Use of PPE is required during the conduct of preparing media. 6. Ensure sufficient quantity.
▪ Dehydrated culture media are highly hygroscopic. Keep container tightly closed 7. Follow recommended time of transport.
once opened. 8. Label specimen accordingly.
▪ Dehydrated culture media are for lab use only. 9. Accompany specimen with complete request form.
▪ Wash hands after every preparation.
▪ Do not eat and use for food (???) CLINICAL SPECIMENS FOR AEROBIC CULTURE
▪ Observe aseptic techniques. Sterile Samples Non-sterile Samples
▪ Do not use culture media after the expiry date. ▪ Blood ▪ Upper respiratory tract spx.
▪ Check the instruction label before storing the culture media. ▪ CSF ▪ Lower respiratory tract spx.
▪ Effusions ▪ Urine
BASIC GUIDELINES ▪ Exudates
Storage:
STERILE SAMPLES
▪ Keep container tightly closed once opened.
1. BLOOD
▪ Do not use culture media after expiry date.
When is a blood culture requested?
▪ Check the instruction label before storing the culture media.
Sterilization: ▪ Acute illnesses ▪ Acute infective endocarditis
▪ Fever of unknown origin ▪ Suspected bacterial
▪ Sterilization by heat is the most used.
endocarditis
▪ Media containing carbohydrates should not be autoclaved at temperature
exceeding 116 deg. C to 118 deg. C.
Components of a Blood Culture Broth:
▪ In some liquid media, warming may lead to loss of activity of some compound. In
▪ 1% Gelatin
these cases, sterilization by filtration must be performed.
o Enhances the growth of N. meningitidis
▪ 0.1% Bacto Agar
1. Weigh an amount of the powder according to the manufacturer’s instruction and o Enhances the growth of anaerobic organism
place in an Erlenmeyer flask. ▪ 0.025% SPS
2. Label the flask with the name of the medium and volume. o Anticoagulant
3. Cover with aluminum foil. o Inhibits the activity of complement and lysosome
4. Autoclave at 121 deg. C for 15 minutes. o Detects phagocytosis
5. After autoclaving, cool to 45-50 deg. C. o Inactivates therapeutic concentration of aminoglycosides
6. For BAP, aseptically add 5-10% sterile defibrinated sheep blood. Allow the
blood to equilibrate at room temperature before adding.
 MEDT 25 – CLINICAL INTERNSHIP 1 (EACMC) 2

Collection Method: 3. URINE

1. Find puncture site. Clean site using 70% alcohol, followed by iodide solution. Methods for Collection:
2. Sterilize the rubber stopper using alcohol swab. a. Midstream Clean Catch
3. Collect the required amount of blood. b. Cytoscopy or Catheterization
4. Inoculate collected blood into the blood culture broth. c. Suprapubic aspiration
5. Label the specimen properly.
4. TRANSUDATES AND EXUDATES
General Rule on Collection:
▪ Clean the site first and disinfect surrounding area before collecting samples.

A. ABSCESS
Collection Method:
▪ General – remove surface exudate by wiping with sterile saline or 70% alcohol.
▪ Open Wound – aspirate or swab deep into the lesion to firmly sample the lesion’s
“fresh border”.
▪ Closed Wound – aspirate

B. EAR/ EYE DISCHARGE

2. CEREBROSPINAL FLUID (CSF)
▪ First (1st) Tube: protein, glucose, and special tests C. TISSUE
▪ Second (2nd) Tube: gram stain and culture ▪ Tissue collection is an invasive procedure and requires surgery by a trained
▪ Third (3rd) Tube: cell count and differential count physician.
▪ Place the specimen in a sterile container on sterile gauze moistened with sterile
3. EFFUSIONS saline.
What and Where to Collect? ▪ Transport to the laboratory. DO NOT REFRIGERATE!
▪ Synovial fluid – from joints ▪ Tissue submitted in formalin is unacceptable for culture.
▪ Pleural fluid – from pleural cavity (space between lungs and inner chest wall)
▪ Peritoneal fluid – from abdominal cavity TRANSPORT TIME
▪ Pericardial fluid – from pericardial cavity Specimen Time Recommended
▪ Hydrocele – from testes Respiratory (i.e., sputum) 1 hour
Gastrointestinal (i.e., stool) 1 hour
NONSTERILE SAMPLES Blood 1 hour
1. UPPER RESPIRATORY TRACT CSF Immediately
Other Body Fluids Immediately
a. Nasopharyngeal Swab
Urine 1 hour
Exudates and Transudates 30 minutes

PLATING TECHNIQUE
A. ISOLATION STREAK TECHNIQUE
1. The inoculation loop is dragged across the surface of the agar back and forth in a
zigzag motion until approximately 30% of the plate has been covered.
2. Sterilize the loop and rotate the plate 90 degrees. Starting in the previously streaked
section, the loop is dragged through it two to three times continuing the zigzag
b. Throat Swab pattern.
3. The procedure is then repeated once more until the fourth quadrant being cautious
not to touch the previously streaked area in order to get isolated colonies.

B. CONTINUOUS STREAKING TECHNIQUES (FOR URINE ONLY)

1. Get the loopful of the urine
aseptically by submerging
vertically only the loop portion
(shaft not included). Inoculate
the BAP by continuous
streaking, spread the urine downward from the first quadrant and streaking up to
2. LOWER RESPIRATORY TRACT the 4th quadrant without changing loop.
a. Sputum
b. Endotracheal aspirate PROCESSING, INOCULATION, STREAKING AND INCUBATION
c. Bronchoalveolar lavage BLOOD (CONVENTIONAL METHOD)
1. Label the blood culture broth (BCB) with the accession number of the sample (e.g.,
Suitability Criteria for Culture: BL001). Incubate for 18-24 hours.
▪ Classification of sputum on the basis of leukocyte and squamous epithelial cell 2. Subculture from BCB is done as follows:
densities. o Label the plates with laboratory number of the sample (e.g., BL001).
o Mix the blood sample by swirling the blood culture bottle 2-3 times.
Cell Numbers per x100 (Low Power) Field
o By using a forceps, get a sterile cotton ball with 70% alcohol and sterilize the
Group Leukocyte Cells Epithelial
6 <25 <25 rubber stopper then gently pass into the flame.
5 >25 <10 o With a sterile disposable syringe and needle, aspirate at least 0.5 mL of blood
4 >25 10-25 from the bottle.
3 >25 >25 o Place a drop of the sample on each agar plate (BAP, CAP, and MAC).
2 10-25 >25 o Streak with isolation.
1 <10 >25 3. Incubate: BAP and CA, in 5-10% CO2 at 36 ± 1 deg. C incubator; MAC at 36 ±
(Only sputum samples in these categories enclosed with brackets ‘[ ]’ should be cultured) 1 deg. C incubator; ambient air for 18-24 hours.
4. Record the culture media plates used and the date and time of inoculation in the
worksheet.
 MEDT 25 – CLINICAL INTERNSHIP 1 (EACMC) 3

5. If no growth, reincubate the BCB until 7 days at 36 ± 1 deg. C; ambient air for 5. Incubate inverted plates: BAP, CA, GBA, and BCA, in CO2 at 36 ± 1 deg. C
further subculture. incubator; MAC at 36 ± 1 deg. C incubator; ambient air for 18-24 hours.
6. Record the culture media plates/ tubes used and the date and time of inoculation
in the worksheet.

CSF/ EFFUSIONS
1. Label the plates and tube with the accession number of the sample (e.g., EX001).
2. About 1 mL preferred (if more than 1 mL is submitted, centrifugation is THOAT SWAB
recommended). Use the sediment (do not decant all fluid but leave at least 1 mL) 1. Label the plates and tubes
for the bacteriological investigation. with accession number of the
3. With a sterile Pasteur pipette, place 1 drop of CSF onto the first quadrant of sample (e.g., R001).
each plate (BAP, MAC, CA) and 3-5 drops of BHI broth. 2. Roll the sides and tip of the
4. Streak with isolation onto each agar plate. swab onto the upper corner of
5. Incubate inverted plates: BAP and CA, in 5-10% CO2 at 36 ± 1 deg. C; ambient the 1st quadrant of BAP.
air for 18-24 hours. 3. Perform isolation streak
6. Record the culture media plates/ tubes used and the date and time of inoculation technique.
in the worksheet. 4. Incubate inverted plates:
CO2 at 36 ± 1 deg. C incubator for 18-24 hours.
5. Record the culture media plates used and the date and time of inoculation in the
worksheet.

URINE
1. Label the plates and tube with the accession number of the sample (e.g., EX001).
2. Mix thoroughly the urine specimen; if sample container is small, invert 3-4 times
or if the sample container is large.
3. Get a loopful of the urine aseptically by submerging vertically only the loop portion
(shaft not included). Inoculate the BAP by continuous streaking, spread the
urine downward from the first quadrant and streaking up to the 4th quadrant without
changing loop.
4. Use another sterile loop and get loopful of urine to inoculate MAC agar and do the
WOUND SWAB/ DISCHARGE isolation streak.
1. Label the plates and tube with accession number of the sample (e.g., EX001). 5. Incubate inverted plates: 36 ± 1 deg. C incubator for 18-24 hours.
2. Use 2 swabs. Using the first swab, roll the sides and tip of the swabs onto the 6. Record the culture media plates used and the date and time of inoculation in the
upper corner of the 1st quadrant of BAP and MAC. worksheet.
3. Submerge the swab in the thioglycolate broth and then aseptically break/ cut the
stick halfway.
4. Perform isolation streak technique onto the BAP and MAC.
5. Incubate inverted plates and thioglycolate broth at 36 ± 1 deg. C for 18-24 hours.
6. Record the culture media plates/ tubes used and the date and time of inoculation
in the worksheet.

STOOL/ RECTAL SWAB

1. Label the plated and tube with accession number of the sample (e.g., S001).
2. Dip a swab into the stool sample.
3. Inoculate the swab onto BAP, MAC, SSA, and TCBS.
4. Inoculate by rubbing the swab onto the center of the first quadrant of one
medium. Do not discard the swab and proceed to the next step.
SPUTUM/ ENDOTRACHEAL ASPIRATE 5. Dip the swab into the upper portion of the APW then into the Selenite F broth.
1. Label the plates with accession number of the sample (e.g., EX001). 6. Streak with isolation onto each agar plate.
2. Get a loopful of purulent part of the specimen to be tested and make an evenly 7. Incubate inverted plates: BAP, MAC, TCBS, and SSA plates, 36 ± 1 deg. C in
thin smear on a slide for gram stain. Air dry. Before sterilizing the loop, dip the loop ambient air for 18-24 hours.
by rubbing in sand alcohol jar to clean the debris left on the loop. 8. Incubate the Enrichment broth media:
3. Using another sterilized loop, get another loopful of purulent part of the o At 36 ± 1 deg. C in ambient air for 6 hours; or
specimen and inoculate each plate with similar size and quality of sample onto the o If the processing of specimen was done in the late office hours and no night
center of the first quadrant. duty, the enrichment broth media should be subcultured after, and
4. Streak with isolation onto each agar plate. overnight of 16-18 hours incubation at room temperature.
9. After incubation, subculture the enrichment broths:

o Label the plates (TCBS, and SSA) with the specimen number and the
source (e.g., S001 AP-TCBS, S001 SF-SSA).
o For APW, aseptically get a loopful of broth at the surface and make an
isolation streak onto TCBS.
✓ Incubate the plates at 36 ± 1 deg. C for 18-24 hours
o For Selenite F, gently mix an get a loopful of broth and make an isolation
streak onto SSA.
✓ Incubate plates at 36 ± 1 deg. C for 18-24 hours
10. Record the culture media plates/ tubes used and the date and time of inoculation
in the worksheet.
REMARKS:
After incubating the enrichment broths:
1. For APW, aseptically get a loopful of broth at the surface and make an isolation
streak onto TCBS.
2. For Selenite F, gently mix and get a loopful of broth and make an isolation
streak onto SSA.
3. Incubate the plates at 36 ± 1 deg. C for 18-24 hours.
Final Report: After 2 days
IDENTIFICATION AND WORK-UPS OF SPECIMEN
SPUTUM/ ENDOTRACHEAL ASPIRATE
▪ After incubation (18-24 hrs), examine the plates (BAP, GBA, BCA, and MAC) for
growth.
NOTE: BAP (Blood Agar Plate), GBA (Gentamycin Blood Agar), BCA (Bacitracin
Chocolate Agar), MAC (MacConkey Agar); Before, they only use BAP, CA, and MAC. But
after their (Ms. Catulin) training in RITM last 2018, GBA and BCA got incorporated in their
sensitivity tests.
▪ GBA – selective media for the isolation of S. pneumoniae. Basically, it is like BAP
with Gentamycin. Gentamycin inhibits the growth of normal flora of the upper
respiratory tract.
▪ BCA – also like chocolate agar, with Bacitracin as its inhibitory substance – which
inhibits the growth of the normal flora of upper respiratory tract. This is a selective
medium for the isolation of Haemophilus influenzae.
1. Growth on BAP:
NOTE: They perform 4 streaks. Quantify…
▪ Light growth – refers to growth on control and primary streak.
▪ Moderate growth – from the primary up to the secondary streak.
▪ Moderately Heavy growth – growth up to the tertiary streak.
▪ Heavy growth – full-plate growth
NOTE: Describe the colony…
▪ α-hemolytic – partial hemolysis (green color around the colony); e.g., S.
pneumoniae, Viridans group, and Enterococcus spp.
▪ β-hemolytic – complete hemolysis (clear hemolysis); e.g., S. pyogenes, S.
agalactiae, S. dysagalactiae, S. equi, Staphylococcus
▪ γ-hemolytic – non-hemolytic; Enterococcaceae, Enterococcus faecalis, E.
faecium, Staphylococcus aureus, and other Staphylococcus species.
MEDT 25 – CLINICAL INTERNSHIP 1 (EACMC) 4
NOTE: When there is growth on BAP, we assume it is Gram (+), but we have to verify it,
since BAP supports the growth of gram (-), gram (+), and yeast. We verify it with gram
staining. Once confirmed that it is Gram-positive cocci, you proceed with catalase test
– uses 3% H2O2, to differentiate Staph from Strep (positive result is effervescence). Once
confirmed as catalase positive, you proceed with coagulase test – which further specify
S. aureus from other Staphs (positive result is coagulation). Once confirmed as coagulase
positive, you proceed with sensitivity testing. If not, we will not consider it as pathogenic
in sputum. If coagulase negative, we report it as “no pathogen isolated”.
NOTE: If in catalase test it tested negative, the work up should be Optochin/ Bacitracin,
depending on the hemolysis – if it is alpha-hemolytic (S. pneumoniae), we perform
Optochin susceptibility testing. In here, a zone of inhibition of >14 mm, indicates S.
pneumonia infection. If the zone of inhibition is <14 mm, we will not proceed to sensitivity
testing, and we will consider it as normal flora; if it is beta-hemolytic, Bacitracin
susceptibility testing for the identification of S. pyogenes. We consider bacitracin sensitive
to S. pyogenes if there is a zone of inhibition, regardless of the size of the zone. Once the
bacteria are sensitive to either Optochin or Bacitracin, that is the only time that we will
proceed to sensitivity testing. Both S. pneumoniae and S. pyogenes are the ones that
we will use for sensitivity testing, and the medium would be Mueller-Hinton Agar (MHA)
with 5-10% sheep’s blood. Remember, alpha resistance to optochin or bacitracin, it is
considered a normal flora and will reported as “no pathogen isolated”.
NOTE: If yeast cells are observed in gram staining, we will perform Germ Tube, wherein
a fresh human serum is used or horse serum (if available). There, the organism will be
emulsified and incubated for 4 hours, and then we will proceed with the identification. Germ
tube positive result is indicative of Candida albicans; and if it is germ tube negative
(since in EACMCC, they do not perform further biochemical tests to identify other Candida
species), they just report it as “Candida spp.”. No sensitivity is performed after this, they
proceed to reporting of result.
2. Growth on BAP and MAC:
▪ Compare BAP and MAC. Check if the colonies are the same and presence of
pigment on each medium. (If other colonies are not the same, look also for other
possible pathogens specific for BAP).
NOTE: Describe the colony…
▪ Lactose Fermenter – PINK colony; e.g., E. coli, K. pneumoniae
▪ Late Lactose Fermenter/ Non-Lactose Fermenter – colorless; e.g., P.
aeruginosa, Acinetobacter baumannii
NOTE: Describe the consistency…
NOTE: If the colony is mixed and not isolated, you will have to restrict it until isolation
occurs; because when you work-up, the colonies must be pure. Once a pure colony is
obtained, you can now proceed to biochemical testing. The work-up for LLF/NLF are those
written on the diagram above, with the addition of oxidase.
NOTE: Once biochemical tests are done, there’s no need to wait for the result, and you
can now proceed to sensitivity testing. When performing sensitivity testing on gram-
negative, the medium used is MHA, and the colony is incubated (length of incubation
depends on the requirement of CLSI per organism). After that, you can now perform
identification, and proceed to the reporting of results.
3. Growth on BCA:
NOTE: The work-up for this is purely for Haemophilus since the growth is on BCA which is
a selective medium for H. influenzae.
NOTE: Describe the morphology – physical appearance of the colony in the agar.
NOTE: In gram staining, what we look for Haemophilus is gram (-) bacilli/ coccobacilli.
The description pleomorphic is always there because Haemophilus exist in different
shapes in gram stain. For the next step, refer to the diagram above. Once confirmed in
the X and V factor dependence test satellitism/ hemolysis test, you will now proceed to
sensitivity testing using Haemophilus Test Medium (HTM). Since Haemophilus is
fastidious during sensitivity testing, when performing other biochemical tests, it should be
incubated under 5-10% CO2, even the sensitivity media. After sensitivity testing, you can
now proceed to identification and reporting of results.
 MEDT 25 – CLINICAL INTERNSHIP 1 (EACMC) 5

4. Growth on BAP/ GBA: 3. Growth on CAP:

NOTE: Since the growth is on GBA, the suspected organism for this is S. pneumoniae, since
GBC is a selective medium for this bacterium.

NOTE: Describe the morphology – Streptococcus pneumoniae is really alpha-hemolytic in 4. No Growth:

BAP and, it is also mucoid.

NOTE: The work-up for S. pneumoniae is the Optochin Susceptibility testing. Then,
for the sensitivity testing, the medium used is MHA with 5-10% sheep’s blood. This also has
to be incubated under 10% CO2.
NOTE: In the preliminary report, it is written “no growth after 24 hours of incubation”.
If after 7 days there is still no growth, the final report will be “no growth after 7 days of
5. No Growth:
incubation”.
NOTE: It is important that when our blood samples are from adults, 2 samples (collected
from different sites) are usually required by the doctor to be collected. It has to be specified
on your result form - the site from where you collected the blood sample.
▪ Final Report: “no growth after 2 days on incubation”
WOUND DISCHARGE/ SWAB
BLOOD ▪ After incubation, examine the primary media plates (BAP, MAC), and thioglycolate
▪ After incubation, examine the primary media plates (BAP, CA, and MAC) for for growth.
growth.
▪ Again, there are no automated machines that is why, the incubation of their blood 1. Growth on BAP:
culture broth is inside the incubator.
▪ Once they receive the bottle, the incubate it right away and after 18-24 hours,
they perform blind subculture – as the name implies, you are not sure/ you do not
know if there is growth. The blood culture bottle that they use in here has color
indicator (grayish-brown) at the bottom, that turns YELLOW when there is
growth. Regardless of the color change of the indicator, subculture in BAP, CAP,
and MAC is still needed, then incubate.

1. Growth on BAP Only:

2. Growth on BAP and MAC:

▪ Compare BAP and MAC. Check if the colonies are the same and presence of
pigment on each medium. (If other colonies are not the same, look also for other
possible pathogens specific for BAP).

NOTE: If catalase positive, proceed with the same work-up with the sputum/ endotracheal
aspirate, but in coagulase test, instead of waiting for the result, you can already proceed
with sensitivity testing right away. Remember, blood is sterile that is why any growth in
there is significant.

NOTE: If it is catalase negative, the work-up is optochin, bile esculin, 6.5% NaCl, and
arabinose (if alpha-hemolytic); bacitracin, CAMP test, bile esculin, 6.5% NaCl, and
arabinose (if beta-hemolytic); and only BE, 6.5% NaCl, and arabinose (if non-hemolytic).
Whatever type of hemolysis is present, BE, 6.5% NaCl, and arabinose must be included
in the work-up because these three are what we used to identify Enterococcus spp., since
this species may occur as alpha, beta, or non-hemolytic.
NOTE: For yeast cells, it has the same work-up with sputum/ endotracheal aspirate.
2. Growth on BAP and MAC:
▪ Compare BAP and MAC. Check if the colonies are the same and presence of
pigment on each medium. (If other colonies are not the same, look also for other
possible pathogens specific for BAP).
3. With Evidence of Growth on THIO, but No Growth on Primary Plates:
NOTE: Thioglycolate broth is an enrichment medium. For example, we did not see any
growth from the primary plates, but the thioglycolate broth became turbid, it has to be
subcultured to BAP and MAC – just get a loopful of the medium and subculture it. After 18-
24 hours, observe for the growth and perform work-up/ sensitivity testing, depending on the
growth observed.
NOTE: if there really is growth, we will put on the final report – “positive for Escherichia
coli”, then we will specify on the final report that it was from the thioglycolate broth. No need
for quantitation.
4. No Growth on Primary Plates and THIO:

NOTE: Preliminary report is released after 24 hours – “no growth after 24 hours of
incubation”. If after reincubation and there is still no growth, the final report should be “no
growth after 4 days of incubation”.

NOTE: If there are other pathogens seen in BAP that are not the same with those in MAC, NOTE: After releasing preliminary report, the primary plates will have to be incubated
perform workup for gram-positive. Then, proceed with the same work-up as that of the for up to 2 days. If after 48 hours there is still no growth on the primary plates, we can
sputum/ endotracheal aspirate. discard those plates, and continue incubation of thioglycolate.
 MEDT 25 – CLINICAL INTERNSHIP 1 (EACMC) 6

CSF/ EFFUSIONS
▪ After incubation, examine the primary media plates (BAP, CAP, and MAC), and
TSB/ BHI for growth.

1. Growth on BAP

3. No Growth:

▪ Final Report: “no growth after 2 days of incubation”

2. Growth on BAP and MAC: STOOL

▪ Compare BAP and MAC. Check if the colonies are the same and presence of ▪ Observe for a typical colony on the following culture media after 18-24 hours of
pigment on each medium. (If other colonies are not the same, look also for other incubation:
possible pathogens specific for BAP). CULTURE MEDIA SUSPECTED COLONIES POSSIBLE ORGANISMS
BAP White β-hemolytic/ non-hemolytic Staphylococcus aureus; yeast
colonies
MAC Non-lactose fermenters or colorless Salmonella spp., Shigella spp.,
(NLF) Aeromonas spp., Plesiomonas spp.,
Escherichia coli (diarrheagenic)
TCBS Yellow or green flat colonies (Y/G) Vibrio cholerae; V. parahemolyticus.
SSA NLF with or without H2S Salmonella spp., Shigella spp.
(H2S-producer colonies on SSA exhibit black coloration on the center, which is a characteristic
of Salmonella. But not all Salmonella are H2S-producers, they can also appear as just colorless,
without black center. Shigella, on the other hand do not really produce black center because it
is not an H2S-producer)

3. Growth on CAP: ▪ Perform biochemical tests/ serological typing on suspected typical colonies
on each medium. Only after this is when we can proceed to sensitivity testing.

BIOCHEMICAL TESTS IDENTIFICATION OF FERMENTERS & NON-FERMENTERS

CITRATE
▪ Used to determine the ability of bacteria to
utilize sodium citrate as its only carbon source
4. With Evidence of Growth on BHI/TSB, but No Growth on Primary Plates: and inorganic ammonium dihydrogen
phosphate (NH4H2PO4) is the sole fixed nitrogen
source.
▪ Make a vertical streak, then fishtail over slant.
▪ Remember, the evidence of growth in BHI/ TSB, and Thioglycolate, is TURBIDITY.

5. No Growth on Primary Plates and BHI/TSB:

TRIPLE SUGAR ION (TSI)
▪ Used to differentiate among the different groups of
Enterobacteriaceae.
▪ Detects 3 primary characteristics of a bacterium:
o The ability to ferment sugars.
URINE
o The ability to produce gas from the fermentation of sugars.
▪ After incubation, examine the plates (BAP, MAC) for growth. Quantitate growth
o Production of large amounts of hydrogen sulfide.
on BAP by multiplying the number of each colony type by 1,000 if 1uL (0.001 mL)
▪ Stab through the center of the butt up to the bottom of the tube then
loop was used or by 100 if 10uL (0.01 mL) loop was used.
draw out and from the lower portion of the slant, make a vertical streak
then fishtail over slant.
1. Growth on BAP:

LYSINE IRON AGAR (LIA)

2. Growth on BAP and MAC:
▪ Compare BAP and MAC. Check if the colonies are the same and presence of
pigment on each medium. (If other colonies are not the same, look also for other
possible pathogens specific for BAP).
▪ Used to determine whether a Gram (-) rod decarboxylates
or deaminates lysine.
o Lysine Deamination – occurs on the lysine slant.
o Lysine Decarboxylation – occurs on the lysine butt.
▪ Stab twice through the center of the butt up to the bottom of
the tube then draw out, and from the lower portion of the slant,
make a vertical streak then fishtail over slant.
 MEDT 25 – CLINICAL INTERNSHIP 1 (EACMC) 7

▪ The OF medium has a low agar and peptone content, and a high sugar content,
making it semi-solid medium that is unlikely to go alkaline from protein utilization if
the sugars are metabolized.
▪ Inoculate the test medium by stabbing it 3x.

IDENTIFICATION OF STAPHYLOCOCCUS SPP.

Staphylococcus spp.
▪ Gram-positive bacteria ▪ Facultative anaerobes
▪ Round/ cocci ▪ Heat-resistant organism
SULFIDE-INDOLE-MOTILITY MEDIUM (SIM) ▪ Grape-like clusters ▪ Can tolerate high salt content-
▪ Non-spore forming bacteria media
▪ A semi-solid medium used as a differential test medium.
o SULFIDE – detection of the ability of an organism to liberate
hydrogen sulfide (H2S) from sulfur-bearing amino acids IDENTIFICATION
producing a visible, black color reaction. 1. Describe the colony morphology on 2. Gram Staining:
o INDOLE – to determine the ability of an organism to split BAP: ▪ Gram (+) cocci
▪ Size ▪ Form clusters “grape-like”
indole from the tryptophan molecule by adding 3-5 drops of
▪ Color ▪ Occur singly, in pairs, tetrads,
Ehrlich’s or Kovac’s reagent and observe for development of
▪ Hemolysis (beta or gamma) short chains
red color.
o MOTILITY – to determine if the organism is motile or non-motile. CATALASE TEST
▪ Stab once through the center of the agar to a depth of 1/3 to 1/2 of the medium ▪ Used to differentiate Staphylococcus (+) from Streptococcus (-).
making sure that the inoculating needle was drawn out in the same stab.
▪ Reagent: hydrogen peroxide (H2O2) – catalase mediates the breakdown of
(NOTE: Indole – performed after incubation of SIM.) H2O2 to hydrogen and water.
▪ After incubation, add 5-7 drops of Ehrlich/ Kovac’s reagent. ▪ Pick a colony from an 18-24-hour culture and immerse into the H2O2. DO NOT
MIX!
▪ Result:
o Positive – immediate bubbling
or effervescence
o Negative – no bubbling formed

COAGULASE TEST
▪ Used to differentiate Staphylococcus aureus (positive) from other
Staphylococcus spp. (negative) – Coagulase Negative Staphylococcus
(CoNS).
▪ Coagulase is an enzyme produced by S. aureus that converts (soluble) fibrinogen
UREASE in plasma to insoluble fibrin.
▪ Used to determine if the microorganism that possesses the enzyme urease
(that can hydrolyze urea, releasing ammonia), and produce a pink-red color SLIDE COAGULASE TEST:
change. ▪ Screening
▪ Inoculate the broth with a heavy inoculum from an 18-24-hour pure culture. ▪ Detects clumping factor (bound coagulase)
▪ Read results: in 10 seconds
▪ Procedure:
o Place 2 drops of reconstituted coagulase plasma on a clean glass slide.
o Pick a colony of the test isolate and emulsify into the drop of coagulase
plasma.
▪ Result:
o Positive – presence of white precipitate or agglutination within 10-15
seconds.
OXIDASE
o Negative – smooth and milky/ homogenous mixture.
▪ Used to determine the presence of
TUBE COAGULASE TEST:
bacterial cytochrome oxidase using the
oxidation of the substrate tetramethyl-p- ▪ Definitive
phenylene dihydrochloride to indophenol – ▪ Detects free coagulase
a dark purple-colored end product. ▪ Reagents: commercially prepared Rabbit’s plasma/ dehydrated plasma containing
▪ It is used to separate citrate or EDTA.
Enterobacteriaceae from other bacteria ▪ Examine tube after 2, 4, & 24 hours of incubation.
like Vibrios, Neisseria spp., ▪ Procedure:
Pseudomonas, Haemophilus, and other o Emulsify 1-3 colonies of the isolate in a tube containing 0.5 mL of
related bacterial species. coagulase plasma.
▪ Place a filter paper on a slide. o Incubate at 36 deg. C for 2-4 hours and observe for a clot formation by
▪ Dispense 1 drop of reagent onto the filter paper. gently tilting the tube.
▪ By using a sterile applicator stick, pick a colony from a colorless media and rub on o If no clot is observed, reincubate the tube and read after 24 hours.
the moistened filter paper. ▪ Result:
o Positive – any degree of clotting/ coagulum
O/F MALTOSE/ DEXTROSE o Negative – no clot/ coagulum formation
▪ Tests the metabolism of sugar by
prokaryotic cells. Cells may
metabolize sugar in a variety of
pathways and the OF test is
studying whether the sugar is
metabolized by aerobic respiration
or by an anaerobic pathway
including fermentation.
 MEDT 25 – CLINICAL INTERNSHIP 1 (EACMC) 8

FLOW DIAGRAM ON IDENTIFICATION OF STAPHYLOCOCCCUS SPP. CAMP TEST

▪ Christie, Atkins, Munch-Petersen
▪ Identify group B (β-hemolytic streptococci) – Streptococcus agalactiae, based
on their formation of a substance called “CAMP factor” that enlarges the area of
hemolysis formed by the β-hemolysin elaborated from S. aureus.
▪ Procedure:
o Make a vertical line using a sterile loop with a few colonies of S. aureus
ATCC 25923 on BAP.
o Draw a perpendicular line of the test isolate
about 4-6 mm away from the S. aureus ATCC
25923 line.
▪ Result:
o Positive – arrow-shaped zone of hemolysis
o Negative – no arrow-shaped hemolysis formed

BILE ESCULIN
▪ Tests the ability of organisms to hydrolyze esculin in the presence of bile. It
IDENTIFICATION OF STREPTOCOCCUS/ ENTEROCOCCUS
is commonly used to identify members of the genus Enterococcus (E. faecalis
Streptococcus spp.
and E. faecium).
▪ Gram-positive cocci in pairs & ▪ 35 deg. C in 5-10% CO2
▪ Procedure:
chains atmosphere
o Inoculate the bile esculin medium with 2-3 colonies of
▪ Non-motile, non-spore forming ▪ Blood, CSF, Upper
▪ Facultative anaerobic Respiratory Tract, LRT, the test isolate with an inoculating needle.
▪ Catalase negative Exudate ▪ Result:
▪ BAP and CAP o Positive – diffuse blackening of more than half of the
slant within 24-4 hours
Streptococcus Classification: o Negative – no blackening
▪ Hemolytic reaction on SBAP
▪ Physiologic – pyogenic, viridans, lactic & enterococcal 6.5% NaCl
▪ Lancefield-Group A-H, L-O ▪ Tests the ability of organisms to grow on high concentrations of salt.
▪ Procedure:
LABORATORY IDENTIFICATION (STREPTOCOCCUS) o Emulsify 2-3 colonies of the test isolate into 6.5%
▪ Bacitracin Susceptibility Test ▪ Bile Esculin NaCl broth.
▪ Optochin Susceptibility Test ▪ 6.5% NaCl ▪ Result:
▪ CAMP Test o Positive – turbidity or presence of obvious
bacterial growth in the medium
BACITRACIN SUSCEPTIBILITY TEST o Negative – clear or no growth
▪ Used to distinguish Group A streptococci, from other streptococci. This test
is used to determine the effect of a small amount of bacitracin (0.04 U) in an ARABINOSE FERMENTATION TEST
organism. ▪ Differentiates Enterococcus faecium (+) from Enterococcus faecalis (-)
▪ Procedure: ▪ Procedure:
o Create a three-layered lawn on half of the BAP with a sterile loop containing o Stab the tubed medium 5-8 times
a few colonies on the test isolate. with 5-10 colonies of the test
o Make sure that the lawn is formed side by side by streaking to ensure isolate.
confluent growth. ▪ Result:
o With sterile forceps, place a bacitracin disk on the lawn. Gently press the o Positive – yellow color change
disk so that it adheres to the agar o Negative – no color change
surface.
o Incubate the plate for 18-24 hours at 36
deg. C, in 5-10% CO2. IDENTIFICATION OF HAEMOPHILUS INFLUENZAE AND NEISSERIA GONORRHOEAE
▪ Result: Haemophilus influenzae
o Susceptible – any zone of inhibition ▪ Large, colorless to gray ▪ Gram-negative coccobacilli
around the disk; Streptococcus colonies ▪ Pleomorphic
pyogenes ▪ No discoloration of CAP ▪ With pungent odor
o Resistant – no zone of inhibition
Growth Requirement of Haemophilus:
OPTOCHIN SUSCEPTIBILITY TEST ▪ X Factor or Haemin
▪ Used in the presumptive identification of alpha-hemolytic Streptococcus ▪ V Factor or NAD (nicotinamide adenine dinucleotide)
pneumoniae, which is optochin sensitive.
▪ Procedure: Identification of Haemophilus:
o With a sterile loop, pick a single alpha- ▪ X and V Factor Dependence Test
hemolytic colony then create a three- ▪ Satellitism/ Hemolysis Test
layered lawn on half of the BAP.
o Make sure that the lawn is formed side by X AND V TEST
side by streaking to ensure confluent ▪ Determines nutrient requirements of organisms
growth. ▪ Procedure:
o Place an optochin disk on the lawn with a sterile forceps. Gently press the o Prepare turbid suspension (~1.0 McFarland)
disk for it to adhere to the agar surface. o Inoculate on trypticase soy agar plate
o Incubate the plate for 18-24 hours at 36 deg. C in 5-10% CO2. o Place disks X, V, and XV.
o Incubate in CO2, +35 to +37 deg. C.
ZONE OF INHIBITION INTERPRETATION
≥ 14 mm Susceptible
9-13 mm Intermediate
< 9 mm Resistant
 MEDT 25 – CLINICAL INTERNSHIP 1 (EACMC) 9

Staining Method: Negative Staining

▪ The background is colored to create a
contrast to aid in the better visualization of
cellular structures.
▪ Example: India Ink

GRAM STAIN
SATELLITISM/ HEMOLYSIS TEST ▪ A differential staining technique based on bacterial cell wall structure.
▪ When grown in blood culture media, S. aureus produce NAD as a metabolic by- ▪ Classifies bacteria as: Gram-positive or Gram-negative
product. For that reason, species of Haemophilus may grow very closely to the
colonies of S. aureus when streaked on sheep blood agar. Gram Stain Reagent:
▪ S. aureus produces NAD (V Factor), a key requirement for the growth of H.
influenzae. This phenomenon is known as “satelliting” and the test is called
“satellitism”.
▪ Procedure:
o Using the same suspension for the X and V growth requirement test, dip the
sterile swab carefully into the bacterial suspension and streak close in a
zigzag motion of BAP. Allow to dry.
o Streak vertical line of S. aureus on the middle of the zigzag pattern and
incubate in a 5-10% CO2 incubator for 1-24 hours at 36+ deg. C.
▪ Result: GRAM STAINING RULES
o Positive – presence of growth with β-hemolysis near or around the S. aureus All COCCI are gram-positive EXCEPT: All BACILLI are gram-negative EXCEPT:
streak ▪ Neisseria ▪ Bifidobaterium
o Negative – presence of growth without β-hemolysis ▪ Veilionella ▪ Streptomyces
▪ Moraxella (Branhamella) ▪ Mycobacterium
▪ Listeria
REMEMBER: ▪ Bacillus
“NeVerMind” ▪ Actinomyces
▪ Clostridium
▪ Corynebacterium
▪ Lactobacillus
▪ Arcanobacterium
▪ Kurthia
▪ Nocardia
▪ Tsukamurella
▪ Propionibacterium
▪ Rothia
Neisseria gonorrhoeae ▪ Erysipelothrix
▪ Grows on BAP (non- ▪ Can survive either as an
REMEMBER:
hemolytic) and CAP extracellular organism or
“BwiSet, MaLa BACCLA Ka Na Talaga
▪ Gram-negative diplococci: alternatively, as an
PRE”
coffee bean-shaped intracellular organism.
Gram Stain Morphology:
IDENTIFICATION OF NEISSERIA GONORRHOEAE
▪ Oxidase Test
▪ Superoxol Test

OXIDASE TEST
▪ Determines the presence of bacterial cytochrome oxidase using the oxidation
of the substrate – tetramethyl-p-phenylene dihydrochloride to indophenol (dark
purple-colored end-product).
▪ N. gonorrhoeae: positive

SUPEROXOL TEST
▪ Catalase
GRAM STAIN – DIRECT FROM SAMPLES
▪ 30% hydrogen peroxide
▪ Result: RESPIRATORY/ SPUTUM SAMPLES
Low Power Field (LPF):
o Positive – effervescence
▪ Leukocytes/ Pus Cells: > or < 25
o Negative – little to no effervescence
▪ Epithelial Cells: > or < 25
STAINING TECHNIQUES
Staining Method: Positive Staining Suitability Criteria of Sputum for Culture:
▪ Simple Stain – a stain which provides color ▪ Classification of sputum based on leukocyte and squamous epithelial cell densities.
contrast but gives same color to all
bacteria and cells. LOW POWER FIELD
Leukocytes/ Pus Cells Epithelial Cells
> 25 > 25
< 25 > 25
▪ Differential Stain – a stain which impart
> 25 < 25
different colors to different bacteria and
< 25 < 25
cells.
(Rows shaded with color green are FIT FOR CULTURE; rows unshaded are unfit)
 MEDT 25 – CLINICAL INTERNSHIP 1 (EACMC) 10

EXUDATES/ URINE/ TISSUE

Pus cells, Epithelial cells/LPF Organisms/OIF Smear check points:
<1/LPF 1+ RARE <1/OIF 1+ RARE ▪ Sputum quality
1-10/LPF 2+ FEW 1-10/OIF 2+ FEW ▪ Staining
11-25/LPF 3+ MODERATE 11-25/OIF 3+ MODERATE ▪ Cleanness
>25/LPF 4+ MANY >25/OIF 4+ MANY ▪ Thickness
(NOTE: Indicate the absence of cells as “NONE”, and of organisms as “NO ORGANISM ▪ Size
SEEN”)
Procedure:
Procedure: 1. Label the slide.
1. Prepare a slide smear. Smear a very thin layer onto the slide, enough to dry 2. Using a wooden applicator stick, pick the purulent particles of the specimen.
completely within a few seconds. 3. Spread the specimen evenly unto the center of the slide by making small,
2. Air dry and heat fix by passing the slide 2-3 times over a flame and allow to cool. circular, coil-type pattern smear.
3. Flood the smear with crystal violet and let stand for 1 minute. 4. Spreading should start on the innermost center to the periphery/ border of the
4. Wash gently with water. Pour off excess water. smear.
5. Flood the smear with Gram’s Iodine and let stand for 1 minute. 5. Air dry and heat fix by passing the slide 2-3 times over a flame and allow it to
6. Wash gently with water. Pour off excess water. cool.
7. Decolorize with acetone-alcohol 5-10 seconds until the alcohol runs most clear. 6. Place fixed slide on the staining rack.
Be careful not to over-decolorize. 7. Pour carbol fuchsin on the smear covering it entirely.
8. Immediately wash with water. 8. Apply enough heat underneath until steam comes off from the stain. (Do not boil or
9. Flood slide with safranin and let stand for 1 minute. dry out)
10. Wash gently with water. Air dry. 9. Leave it for 10 minutes.
11. Examine under LPO and estimate number of squamous epithelial cells and 10. Gently rinse with tap water to remove excess carbol fuchsin. At this point, the smear
leukocytes. is red in color.
12. Examine under OIO to determine predominant and other organisms present. 11. Decolorize with acid alcohol until pink color disappears from the smear.
12. Counterstain with
GRAM STAIN – QUALITY CONTROL methylene blue for 2-3
▪ Controls should be run on a daily basis. seconds.
▪ Standard color depends on the manufacturer, as long as Gram-positive is 13. Gently wash with tap water
recognizable from Gram-negative. and tip to drain excess
water.
GRAM STAIN – IMPORTANT POINTS
▪ Use fresh/ young cultures – must be within 18-24 hours. REPORTING SCALE AFB SEEN
▪ Thin, thick, or uneven smears will result in poor/ uneven staining and 0 No AFB seen in 300 visual fields (VF)
decolorization. +n 1-9 AFB seen in 100 VF (write the actual number of AFB seen;
▪ Do not heat-fix for very long. e.g., +1, +2…+9)
▪ Perform quality control of reagents. 1+ 10-99 AFB seen in 100 VF
2+ 1-10 AFB/ OIF in at least 50 VF
INDIA INK 3+ More than 10 AFB/ OIF in at least 20 VF
▪ Staining of CSF to test for the presence of
Cryptococcus spp. OTHER TESTS:
POTASSIUM HYDROXIDE (KOH) MOUNT
Procedure: ▪ Used for rapid detection of fungal elements in clinical specimen, as it clears
1. Place a drop of india ink on a clean glass slide. the specimen making fungal elements more visible during direct microscopic
2. Add a drop of sample to the india ink. examination.
3. Mix using the edge of a cover slip. ▪ Reagent: 10% KOH
4. Cover the entire smear with the cover slip.
5. Let it stand for 5 minutes. Procedure:
6. Look for white cells in LPO, then shift to HPO. 1. Place 1 to 2 drops of 10% KOH on a clean glass slide.
2. Add a small amount of specimen in 10% KOH.
Results: 3. Mix the KOH and specimen using the edge of the cover slip.
Findings Reporting 4. Cover the drips with the cover slip.
Encapsulated yeast cells = PRESENT Positive for encapsulated yeast cell/s 5. Wait for 5 minutes for cellular clearing.
Encapsulated yeast cells = ABSENT No encapsulated yeast cell/s seen 6. Examine the slide under the microscope with a reduced light source at LPO.

Observe for the following structures:

ACID-FAST BACILLI STAINING ▪ Yeast cells (budding or single cells)
Acid-Fast Bacilli ▪ Pseudohyphae
▪ Rod-shaped bacilli that can be seen ▪ Hyphal Elements: maybe hyaline (light or no pigment) or dematiaceous (dark
under the microscope following a brown).
staining procedure in which the bacteria
retain the color of the stain after an acid REPORTING
wash. POSITIVE Report as “positive for (fungal elements observed)”;
e.g., Yeast and Hyphal elements
DIRECT SPUTUM SMEAR MICROSCOPY (DSSM) NEGATIVE Report as “no fungal elements found”
▪ Involves the examination of series of sputum specimens from each patient and
requires repeated patient visits to health facilities to submit specimens and to KOH STRING TEST
collect. ▪ Relies on the differential resistance to 3% potassium hydroxide between
▪ Screening for pulmonary tuberculosis. gram positive to negative cells.

Pulmonary Specimen (2 samples): Procedure:

▪ Spot-early morning collection 1. Place a drop of 10% KOH on a clean glass slide.
▪ Spot-spot collection (at least 1-hour apart) 2. Emulsify a loopful of growth from a colony to the drop of 3% KOH.
 MEDT 25 – CLINICAL INTERNSHIP 1 (EACMC) 11

3. Stir the suspension continuously for 60 seconds. DISK DIFFUSION METHOD

4. Gently pull away the loop from the suspension. ▪ Antibiotic paper disks are placed on agar medium surface inoculated with the test
organism.
REPORTING ▪ Kirby-Bauer Method
POSITIVE Organisms become thick, stringy and form long strands within
the first 30 seconds. This is seen in gram negative bacteria. MEDIUM FOR DISK DIFFUSION
NEGATIVE Organisms leave the suspension unaltered or absence of Agar for AST:
stringing. This is seen in gram positive bacteria. ▪ pH: 7.2-7.4
▪ Low Thymine and Thymidine
GERM TUBE ▪ Sufficient divalent cation (Ca2+, Mg2+)
▪ An outgrowth produced by spore-releasing fungi during germination. The germ
tube differentiates, grows and develops by mitosis to create somatic hyphae. Mueller-Hinton Agar (MHA):
▪ Good batch to batch reproducibility.
Procedure: ▪ Low in antibiotic inhibitors against sulfonamides, trimethoprim, and tetracycline.
1. Place 0.5 mL of fresh serum in clean test tube. ▪ Supports the growth of most non-fastidious pathogens.
2. Pick a colony, then emulsify in the fresh serum.
3. Incubate the suspension at 35-37 deg. C for 2-3 hours. AST AGARS
4. After incubation, place a drop of suspension on a clean glass slide and cover with Organism Agar Plate
a cover slip. Gram-negative organisms: Mueller-Hinton Agar (MHA)
5. Examine under LPO and HPO. Staphylococcus spp., Enterococcus spp.
Streptococcus spp., Neisseria meningitidis Mueller-Hinton Agar with 5% sheep’s blood
INTERPRETATION Haemophilus spp. Haemophilus test Medium (HTM)
POSITIVE • A short hyphal (filamentous) extension arising laterally from a Neisseria gonorrhoeae GC Agar
yeast cell, with no constriction at the point of origin.
• Germ tube is half the width and 3-4 times the length of the yeast cell, ANTIBIOTIC DISKS
and there is no presence of nucleus.
▪ Stock antibiotic disks should be stored at -20 deg. C.
NEGATIVE No hyphal (filamentous) extension arising from a yeast cell or a short
▪ Working antibiotic disks should be stored at 4 deg. C.
hyphal extension constricted at the point of origin.
▪ Antibiotic disks used are based on Clinical and Laboratory Standards Institute
SEROTYPING (CLSI) panels.
▪ Serotypes are groups within a single species of microorganisms such as bacteria
or viruses, which share distinctive surface structures. DISK PER PLATE GUIDE
Maximum Number of Disks
Organisms 140 mm or 150 mm (big 100 mm or 90 mm
Procedure:
plate) (small plate)
1. Place a drop of each of anti-sera on a clean glass slide.
• Enterobacteriaceae,
2. Using a sterile applicator stick, pick several colonies from the plate and emulsify
• Pseudomonas aeruginosa,
each drop, creating a homogenous, 12 5
• Acinebacter spp.,
slightly milky suspensions. • Enterococcus spp.
3. Mix the antiserum with the suspension Haemophilus spp. 9 4
using a sterile applicator stick. • Streptococcus pneumoniae,
4. Tilt the glass slide back and forth for 1 • Streptococcus spp. (Viridans
minute and observe for agglutination. group), 9 4
5. Interpret as follows: • Streptococcus spp. (B-
hemolytic group)
INTERPRETATION Neisseria gonorrhoeae 9 3
POSITIVE Strong agglutination appears within 30 seconds to 1 minute. Neisseria meningitidis 5 2
NEGATIVE Homogenous suspension
▪ Agglutination is grossly observed with light passing through the slide. Delayed or INOCULATION OF AGAR PLATES
weak agglutination is regarded as negative. ▪ Use appropriate check plate for particular isolate.

Organism Check Plate

ANTIMICROBIAL SUSCEPTIBILITY TESTING Gram-positive:
▪ Measures the ability of microbial agent/s to inhibit in-vitro bacterial growth. Staphylococcus spp., Streptococcus spp., BAP
▪ Indicated for an etiologic agent of an infection that needs chemotherapy. Neisseria meningitidis
▪ Guide the clinician in selecting the best and appropriate antimicrobial agent (to Gram-negative MAC
predict the outcome of treatment with the antimicrobial tested). Fastidious:
CAP
Haemophilus spp., Neisseria gonorrhoeae
Methods:
▪ Disk diffusion
▪ Dilution method INCUBATION GUIDE
▪ Gradient method ▪ Inverted plates are incubated at the desired temperature, atmosphere, and
▪ Automated Antimicrobial Susceptibility Testing System duration of incubation, depending on the test organism’s requirement.

Minimal Inhibitory Concentration (MIC): Organism Agar Temp. Atmosphere Time

▪ Lowest concentration of an antimicrobial agent that would inhibit visible in-vitro Enterobacteriaceae
growth of a test organism over a defined interval related to the organism’s growth Pseudomonas 16-20 hrs
aeruginosa
rate.
Acinebacter spp.
▪ Determined by:
Burkholderia spp. MHA 35 deg. C ± 2 --
o Dilution Method 20-24 hrs
Stenotrophomonas
o Gradient Diffusion Method
maltophilia
o Automated Antimicrobial Susceptibility Testing System Other non-
16-20 hrs
Enterobacteriaceae
 MEDT 25 – CLINICAL INTERNSHIP 1 (EACMC) 12

Staphylococcus Discrete Colonies Growing within the Zone:

spp. 24 hrs ▪ When discrete colonies grow
Enterococcus spp. within a clear zone of
Streptococcus spp. MHA with inhibition, repeat the test
(B-hemolytic group 5% sheep’s 5% CO2 20-24 hrs with a pure culture or
and Viridans group) blood subculture of a single
colony from the primary
15-MINUTE RULE: culture plate.
Within 15 minutes… ▪ When the discrete colonies continue to grow within the zone of inhibition after
▪ After preparing inoculums, seed the agar repeating the test, measure the colony-free inner zone.
▪ Apply disk on seeded agar
Measuring Zones of Inhibition Using Transmitted Light*:
▪ After disk application, incubate plate ▪ For some tests (e.g., linezolid against Staphylococcus spp.), measure zones of
complete inhibition with transmitted light.
Procedure: ▪ Position the inverted Petri plate in front of a light source for zone
1. Label the MH plate with the specimen accession number. examination.
2. Prepare 2-3 mL sterile NSS in sterile 5 mL round bottom tube.
3. Using a sterilized inoculating loop, select and pick 3-5 similar isolated colonies by Trimethoprim-sulfamethoxazole:
touching the top of the colonies. ▪ When measuring the zone of
4. Dip the loop into NSS and emulsify on the inside wall of the tube for equal inhibition for trimethoprim-
distribution of the organism. sulfamethoxazole,
5. Mix the bacterial suspension by tapping or inverting the tube disregard slight or hazy
6. Using a densitometer, adjust the bacterial suspension to the required 0.5 growth (20% or less of the lawn of growth), and measure the obvious zone
McFarland standard. margin.
7. Dip a sterile cotton swab into the standardized bacterial suspension and express
Swarming:
excess fluid against the inside wall of the tube. ▪ Strains of Proteus spp. may swarm into
8. Swab the entire surface of the AST medium three times, rotating the plate through areas of inhibited growth. Ignore the thin
an angle of about 60 degrees after each application and pass the swab around the veil of swarming growth in an otherwise
rim of the agar surface. obvious zone of inhibition.
9. With the same swab, inoculate by touching/ rubbing the check plate. Discard the
swab accordingly. Double Zones:
10. Streak the inoculum in the check plate with sterile inoculating loop. ▪ When double zones are observed, check
11. Apply appropriate antibiotic disks manually by using sterile forceps onto the the growth for purity and repeat the test,
inoculated agar surface with a minimum spacing of 24 mm center to center between if necessary.
disks. ▪ If the culture is pure, measure the inner
12. Gently press each disk down with sterile forceps for every application of the disk. zone.

READING ZONES QUALITY CONTROL

**Within 15 minutes of applying disks, incubate the inverted ▪ Antibiotics should be regularly tested if they still function properly.
plates** ▪ Guide to which antibiotic to test, with what organism, and their range are
▪ Using calipers or ruler, measure the diameter of the indicated in the CLSI Document no. M100.
complete zone of inhibition. ▪ Control strains MUST be used as a reference:
▪ Read MHA plates with unaided eye using transmitted o Staphylococcus aureus 25923
light. o Escherichia coli 25922
▪ On MHB, GC Agar, and HTM, remove the cover and o Pseudomonas aeruginosa 27853
measure inhibition zones from the surface illuminated o Streptococcus pneumoniae 49619
with reflected light. o Haemophilus influenzae 49247
o Escherichia coli 35218

1. Assessing Growth DETECTION OF MULTIPLE DRUG-RESISTANT ORGANISMS (MDRO)

▪ Read plates only when the lawn of growth is
confluent (A).
▪ Repeat the test when individual colonies are
apparent (B).
MULTIDRUG RESISTANT ORGANISM (MDRO)
▪ This refers to bacteria that have become resistant to more than one class of
antimicrobial agents.
▪ Usually, they are resistant to all but one or two more commercially available
antimicrobial agents, complicating treatment of illnesses they cause.

TEST RECOMMENDATIONS FOR DIFFERENT ORGANISMS

2. Measuring the Zones of Inhibition I. Direct β-Lactamase Test (Nitrocefin Disk) for:
▪ Measure zones of inhibition to the a. Enterococcus spp. d. Neisseria gonorrhoeae
nearest whole millimeter (mm). b. Haemophilus influenzae/ e. Neisseria meningitidis
▪ Measure growth with no zone of parainfluenzae f. Staphylococcus spp.
inhibition as 6 mm (B). c. Moraxella catarrhalis g. Pasteurella spp.
▪ Zones of complete inhibition II. Screening for Staphylococcus spp.
include the diameter of the disk and a. Direct β-Lactamase Test
show no obvious, visible growth as judged by the unaided eye. b. mec-A-mediated Oxacillin Resistance using Cefoxitin
▪ Measure the zone of growth c. Inducible Clindamycin Resistance
inhibition, not the zone of hemolysis. III. For Inducible Clindamycin Resistance (ICR)
Tilt the plate to better differentiate a. Staphylococcus spp.
between hemolysis and growth. b. β-Hemolytic Streptococcus spp.
c. Streptococcus pneumoniae
 MEDT 25 – CLINICAL INTERNSHIP 1 (EACMC) 13

TEST RECOMMENDATIONS FOR ENTEROBACTERIACEAE Interpretation:

I. Extended Spectrum β-Lactamase Test (ESBL) Positive:
▪ (E. coli/ Klebsiella spp./ P. mirabilis) o Flattening (D-zone) of clindamycin zone
II. AmpC β-Lactamase Screening Test adjacent to erythromycin disk or hazy
▪ Not yet recommended by CLSI growth inside the zone of inhibition (no D-
III. Test for Diminished Fluoroquinolone Susceptibility using Pefloxacin (extraintestinal zone) adjacent to erythromycin disk.
Salmonella) Negative:
o No flattening or hazy growth of clindamycin zone adjacent to erythromycin
IV. Carbapenemase Screening Test disk.

V. Metallo-β-Lactamase Test (MBL) EXTENDED SPECTRUM β-LACTAMASES (ESBL)

▪ Plasmid-mediated enzymes produced by Enterobacteriaceae derived from
mutations of TEM-1, TEM-2, and SHV-1
METHICILIN-RESISTANT STAPHYLOCUCCUS AUREUS (MRSA) ▪ Capable of hydrolyzing extended spectrum cephalosporins, penicillins, and
▪ mecA or mecC-mediated oxacillin resistance in Staphylococci aztreonam.
▪ Disk Diffusion: CEFOXITIN as surrogate antibiotic ▪ Most often associated with:
▪ Medium: o Escherichia coli
o Mueller Hinton Agar with 4% NaCl for oxacillin only. o Klebsiella spp.
o Mueller Hinton Agar without NaCl for cefoxitin and other antibiotics. NOTE: Routine screening for Proteus mirabilis for ESBL production is NOT
RECOMMENDED. However, when it is deemed clinically relevant (e.g., Bacteremic
Procedure: isolate), screen for ESBL.
1. Following the standard disk diffusion, place cefoxitin disk onto plain MHA.
(MIC: Oxacillin onto MHA with 2-4% NaCl) ▪ Can be transferred to enteric bacilli (Salmonella, Shigella, Citrobacter freundii,
*MIC = Minimum Inhibitory Concentration and Serratia marcescens).
2. Incubate plain MHA (cefoxitin) for 16-20 hours. ▪ Appear to be susceptible to certain antibiotics.
3. Read susceptibility results. ▪ ESBL-producing isolates are susceptible to Beta-lactam/ Beta-lactamase inhibitor
4. NOTE: for oxacillin (and Vancomycin), read with transmitted light; for cefoxitin combination agents in-vitro, but the effectiveness is uncertain.
(and other antibiotics), read with reflected light. o E.g., Clavulanic acid, Sulbactam, and Tazobactam – inhibit the ESBLs
5. Record and interpret according to the latest CLSI document. produced by the organisms when tested with cefotaxime, ceftazidime,
cefepime or aztreonam.
Cefoxitin Oxacillin Oxacillin
mecA-mediated Resitance Interpretation I. Double Disk Diffusion Method
Disk MIC Report
mecA-mediated (or the rare R R R MRSA ▪ Detection of ESBL is by using a disk containing
mecC) (or do not clavulanic acid (AMC) and placing it between
report) cefotaxime (CTX) and aztreonam (ATM) disk.
mecA-homologues (e.g., R R R MRS ▪ An elliptical clearing of distortion of inhibition
mecA1, mecA2, mecC1) (or do not (other CoNS between the 2 disks indicates an inhibition of β-
report) spp.) Lactamases by clavulanic acid.
(--) S R R Not ▪ Screening Test
MRSA/MRS
(--) S S S MSSA/MSS II. AmpC β-Lactamases
▪ Chromosomal or plasmid-encoded enzymes. Isolates that produce AmpC
PENICILLIN DISK ZONE EDGE TEST FOR β-LACTAMASE PRODUCTION OF STAPH. SPP enzymes may have an antimicrobial susceptibility profile similar to those that
▪ Performed on Mueller-Hinton Agar produce ESBLs in that they show reduced susceptibility to penicillins,
using a 10-U penicillin disk following cephalosporins, and aztreonam.
CLSI guidelines. ▪ Found in Enterobacter spp., K. aerogenes, Citrobacter spp., Serratia spp., and
▪ After 16-18 hours of incubation in some other gram-negative species.
ambient air, a sharp zone edge was
interpreted as positive and a fuzzy Procedure:
zone as negative for β-Lactamase production. 1. Following the general procedure for antimicrobial susceptibility testing, place
cefotaxime (30 ug) disk 15-25 mm apart from edge of imipenem (10ug) disk
β-Lactamase Test Penicillin Disk Penicillin on the susceptibility plate.
Penicillin Disk
REPORT Zone Edge Test REPORT 2. Incubate plates for 16-24 hours.
R (+) Sharp R
3. Read susceptibility test results and observe presence of phenotypic resistance.
S (--) fuzzy S
>29 mm
Interpretation:
S (+) sharp R
>29 mm Positive:
o Flattened edge of the inhibitory zone around
D-TEST: INDUCIBLE CLINDAMYCIN RESISTANCE (ICR) cefotaxime disk adjacent to an imipenem disk.
▪ A disk diffusion test using clindamycin and erythromycin disks placed in Negative:
close proximity to detect the presence of inducible clindamycin resistance in o No flattened edge of the inhibitory zone around
Staphylococci and Streptococci. cefotaxime disk adjacent to an imipenem disk.

Procedure: III. Carbapenemase

1. Following the general procedure for antimicrobial susceptibility testing, place
clindamycin 2ug disk *15-25 mm (Staphylococcus spp.) apart from edge of
erythromycin 15ug disk on the susceptibility plate.
o Beta-hemolytic Streptococcus spp. (12-15 mm disk distance)
o Streptococcus pneumoniae (12-15 mm disk distance)
2. Incubate plates for 16-24 hours.
3. Read susceptibility test results and observe presence of ICR.
▪ β-Lactamases with versatile hydrolytic capacities. They have the ability to
hydrolyze penicillins, cephalosporins, monobactams, and carbapenems. Bacteria
producing these β-lactamases may cause serious infections in which the
carbapenemase activity renders many β-lactams ineffective.
▪ Carbapenemase activity in Enterobacteriaceae and P. aeruginosa can be
confirmed using the CarbaNP colorimetric microtube assay or the modified
carbapenem inactivation method (mCIM) test.
▪ Both CarbaNP and mCIM tests may detect carbapenemase production, but neither
of these tests can identify which carbapenemase is present.

Procedure:
1. For each isolate to be tested, emulsify a 1-uL loopful of bacteria for
Enterobacteriaceae or 10-uL loopful of bacteria for P. aeruginosa from an
overnight blood agar plate in 2 mL TSB.
2. Vortex or mix by inversion for 10-15 seconds.
3. Add a 10-ug meropenem disk to each tube using sterile forceps or a single disk
dispenser. Ensure the entire disk is immersed in the suspension.
4. Incubate at 35 def. C ± 2 deg. C in ambient air for 4 hours ± 15 minutes.
5. Just before or immediately following completion of the TSB-meropenem disk
suspension incubation, prepare a 0.5 McFarland suspension (using the colony
suspension method) of E. coli ATCC 25922 in nutrient broth or saline.
6. Inoculate an MHA plate with E. coli 25922 as for the routine disk diffusion
procedure making sure the inoculum suspension preparation and MHIA plate
inoculation steps are each completed within 15 minutes. Allow the plates to dry for
3-10 minutes before adding the meropenem disks.
7. Remove the meropenem disk from TSB-meropenem disk suspension using a 10-
uL loop by placing the flat side of the loop against the flat edge of the disk and
using surface tension to pull the disk out of the liquid. Carefully drag and press the
loop along the inside edge of the tube to expel excess liquid from the disk. Continue
using the loop to remove the disk from the tube and then place it on the MHA plate
previously inoculated with the meropenem-susceptible E. col 25922 indicator strain.
Disk capacity: 4 disks on a 100 mm MHA plate; 8 disks on a 150 mm MHA
plate.
8. Invert and incubate the MHA plate at 35 deg. C ± 2 deg. C in ambient air for 18-24
hours.
9. Following incubation, measure the zones of inhibition as for the routine disk
diffusion method.
Interpretation:
Carbapenemase Positive:
o Zone diameter of 6-15 mm or presence of pinpoint colonies within a 16-
18 mm zone.
o If the test isolate produces a carbapenemase, the meropenem in the disk
will be hydrolyzed and there will be no inhibition or limited growth
inhibition of the meropenem-susceptible E. coli ATCC 25922.
Carbapenemase Negative:
o Zone diameter of ≥ 19 mm (clear zone).
o If the test isolate does not produce carbapenemase, the meropenem in the
disk will not be hydrolyzed and will inhibit growth of the meropenem-
susceptible E. coli ATCC 25922.
Carbapenemase Indeterminate:
o Zone diameter of 16-18 mm.
o Zone diameter of ≥ 19 mm and the presence of pinpoint colonies within
the zone.
o The presence or absence of carbapenemase cannot be confirmed.
REPORTING OF CULTURE RESULTS
BLOOD
1. Preliminary Report
▪ The preliminary report for the absence of growth should be reported as: “NO
GROWTH AFTER 24 HOURS OF INCUBATION”
2. Final Report
▪ Final report with presence of growth should be reported as: “POSITIVE FOR
(IDENTIFIED ORGANISM) AFTER (HOURS/DAYS) OF INCUBATION”.
(Susceptibility test results included, if applicable).
▪ Final report with negative results for any organism should be reported as: “NO
GROWTH AFTER 7 DAYS OF INCUBATION”
3. Record both preliminary and final report, and date of release in the designated logbook.
Clinical Significance:
▪ If a typical pathogen is found from blood culture, it is almost always
significant. But many bacteria are often significant but may occur as
contaminants in blood culture. Therefore, the finding maybe discussed with the
clinician or place a note in the result form – “Please correlate clinically”.
CSF/ EFFUSIONS
Culture Examinations:
▪ Absence of colonies on the preliminary plates (direct inoculation of specimen
onto agar plates) and BHI broth has no sign of growth, record and report.
Primary plates and BHI broth are inspected everyday for the sign of growth.
▪ Re-incubate all “No Growth” plates up to 2 days (after reading all the plates,
re-incubate).
MEDT 25 – CLINICAL INTERNSHIP 1 (EACMC) 14
▪ Perform subculture if BHI broth has sign of growth while there is no growth
on primary plates.
▪ If primary plates and BHI broth are positive, correlate the organisms
isolated.
1. Preliminary Report
▪ The preliminary report for the absence of growth should be reported as: “NO
GROWTH AFTER 24 HOURS OF INCUBATION”
2. Final Report
▪ Final report with presence of growth on both primary plates and BHI should be
reported as: “POSITIVE FOR (IDENTIFIED ORGANISM” (Susceptibility test
results included, if applicable).
▪ Final report with presence of growth on BHI only should be reported as: “NO
GROWTH ON PRIMARY ISOLATION. POSITIVE FOR (IDENTIFIED
ORGANISM) (FROM BHI BROTH AFTER DAYS OF INCUBATION)”
(Susceptibility test results included, if applicable).
▪ Final report with negative results for any organism should be reported as: “NO
GROWTH AFTER 4 DAYS OF INCUBATION”
3. Record both preliminary and final report, and date of release in the designated logbook.
Clinical Significance:
▪ If a typical pathogen is found from CSF culture, it is almost always
significant. But many bacteria are often significant but may occur as
contaminants in CSF culture. Therefore, the finding maybe discussed with the
clinician or place a note in the result form – “Please correlate clinically”.
EXUDATES
1. Preliminary Report
▪ The preliminary report for the absence of growth should be reported as: “NO
GROWTH AFTER 24 HOURS OF INCUBATION”.
2. Final Report
▪ Final report with presence of growth on both preliminary plates and
thioglycolate should be reported as: “POSITIVE FOR (IDENTIFIED
ORGANISM)” (Susceptibility test results included, if applicable).
▪ Final report with presence of growth on thioglycolate only should be reported
as: “NO GROWTH ON PRIMARY ISOLATION. POSITIVE FOR (IDENTIFIED
ORGANISM) (FROM THIOGLYCOLATE BROTH AFTER DAYS OF
INCUBATION)” (Susceptibility test results included, if applicable).
▪ Final report with negative results for any organism should be reported as: “NO
GROWTH AFTER 4 DAYS OF INCUBATION”
3. Record both preliminary and final report, and date of release in the designated logbook.
URINE
1. Preliminary report is not routinely done after 24 hours of incubation for the presence
and absence of growth.
2. Three or >3 different colonies should be considered and reported as “MIXED
CULTURE” and suggest a “REPEAT COLLECTION”.
3. Three or >3 different colonies should be considered if the patient is an emergency case
or in catheter. Identify and perform susceptibility testing on predominant organisms and
the colony count is >100,000.
4. Final Report
▪ Presence of Growth: “(QUANTITY) CFU OF (IDENTIFIED ORGANISM) PER
ML OF URINE” (Susceptibility test results included, if applicable).
▪ Absence of Growth: “NO GROWTH AFTER 48 HOURS OF INCUBATION”
5. Record final report and date of release in the designated logbook.
STOOL
1. Preliminary report is not routinely done.
2. Final Report
▪ Final report with presence of growth on both primary plates and BHI should be
reported as: “POSITIVE FOR (IDENTIFIED ORGANISM)”.
▪ Stool specimen is known to have normal bacterial fecal flora. If no suspected
pathogens observed or tested, report: “NO IMPORTANT
ENTEROPATHOGEN ISOLATED”.
▪ If no colonies found in culture, report: “NO GROWTH AFTER 2 DAYS OF
INCUBATION”
3. Record final report and date of release in the designated logbook.
 MEDT 25 – CLINICAL INTERNSHIP 1 (EACMC) 15

Q&A:
Q: Have you experienced isolating a multidrug-resistant bacterium, and what is the SOP
for that?
- Whenever we experience this, we report it on the infection control committee. We
still release results in the nursing station, but we also release a copy on the infection
control nurse so that it can be disinfected, and the nurse can be instructed on how
to handle the patient. We have a logbook where we document the communication
regarding that matter.

Q: In the phenotypic test for methicillin-resistant Staphylococcus aureus (MRSA), if it is

resistant to oxacillin, are you going to proceed to cefoxitin sensitivity? How is the MIC of
cefoxitin conducted – how is it measured to confirm the phenotypic test?
- Actually, in MRSA, you can identify it already in the disk diffusion. There, cefoxitin
is used for the identification. Cefoxitin will serve as the surrogate antibiotic. Once it
is resistant to cefoxitin, we will right away report it as methicillin-resistant. There’s
no need to perform MIC, disk diffusion alone is already enough. But, if you have
available procedure in the lab for MIC wherein you will use oxacillin, it is fine as
well. The medium to be used, however, should be MHA with 4% NaCl; and then we
will also base its measurement on the CLSI guidelines.

Q: Regarding KOH, for example the patient applied lotion and ointments on the site, what
will you do to the patient? In antibiotic testing, how far is the recommended distance
when putting them onto the agar?
- For example, the patient applied ointment, what we do is we clean the site or area
applied with ointment. Actually, we are supposed to instruct the patients to not apply
anything onto their skin before collection.
- The distance should be at least 21 mm from each other. In a 90-millimeter plate,
there has to be only 4 antibiotic disks; and for 150-millimeter plates, there has to
be at least 12 antibiotic disks. We cannot exceed this amount because it may
overlap which can affect the reading.

Q: When you isolate something resembling Neisseria from chocolate agar, do you
proceed with biochemical testing (aside from oxidase, catalase, and tributyrin) to
determine the specific species of Neisseria? What is the manner of reporting?
- In EACMCC, we perform gram stain, oxidase, catalase, and sugars. And when
performing sensitivity testing, there is a specific agar used – GC Agar. It is also
incubated under CO2.
- Once identified in the biochemical testing, you report the specific species identified.