LABORATORY MANUAL

MEDICAL MICROBIOLOGY

MIC341
EXPERIMENT 1: ANTIMICROBIAL SUSCEPTIBILITY TESTING

Objectives: After this exercise, the student should be able to:

(A) Test a clinically isolated for susceptibility to antimicrobial using the disk
diffusion assay and the MIC assay.
(B) Read and interpret antimicrobial test and report appropriately.

Introduction:

The final step in processing a clinical specimen is to determine what antibiotic can kill
the infecting organism. This information is necessary for the doctor to recommend the correct
drug to be given. Antimicrobial susceptibility testing (AST) must be conducted using pure
cultures only, or the results must be invalid. There are two ways to do an AST : the Bauer-
Kirby disk diffusion assay or the classical Minimum Inhibitory Concentration (MIC) assay.
The MIC test provides more information regarding the concentration of antibiotic required,
but is more difficult to perform. The disk diffusion test is easier to perform but is suitable only
for aerobic bacteria. It is routinely used in clinical labs.

In the disk diffusion assay, bacteria to be tested is spread evenly onto an agar plate. A
disk containing specific amounts of an antibiotic is then placed onto the lawn and incubated
overnight. The inability of the test bacteria to grow around a particular disk (forming an
inhibition zone) indicates susceptibility to that antibiotic. The diameter of the inhibition zone
is measured and compared to a standard chart. In the MIC test, a series of twofold dilutions of
antibiotic is prepared. The test organism is then inoculated into each concentration, and
growth is checked by observing for turbidity. The highest concentration which no growth is
observed is taken as the MIC.

Material :

• 2x 96 well sterile Microtiter plate

• Multichannel micropippettor
• Micropippete tips yellow
• Barium chloride solution 1.175% (wt/Vol)
• Sulfuric acid 1% (Vol/Vol)
• Nutrient agar
• 1 bottle 24 hours culture of E.coli in Mueller-Hinton Broth
• 1 bottle 24 hours culture of B.subtilis in Mueller-Hinton Broth
• 1 bottle 24 hours culture of S.aureus in Mueller-Hinton broth
• 1 bottle 24 hours culture of P.aeruginosa in Mueller-Hinton broth
• 1 bottle of Nutrient broth
• 4 sterile pasteur pipettes
• 4 sterile cotton swabs
• Pairs of sterile forceps
• 6 or more types of antibiotic disks
Procedures

Each group will test all the 4 bacteria for their antibiotic susceptibility. Each member of the
group will work with one bacteria.

(A) Preparing a 0.5 McFarland turbidity standard

1. Add 0.5ml of 0.048 M barium chloride (1.175%,wt/Vol) to 99.5 ml of 0.36 N Sulfuric

acid (1%, Vol/Vol)
(B) Preparing the standardised inoculum

Step 1 & 2 has been done for you. Start at step 3.

1. Pick 4 or 5 well isolated colonies and inoculate into 5 ml of Mueller-Hinton broth.

Mix.
2. Incubate at 37 ̊ C until the turbidity is about the same as the 0.5 McFarland standard
(usually takes 2 to 8 hours).
3. Compare the turbidity of the culture and the 0.5 McFarland standard by holding both
tubes in front of the white paper with finely drawn black lines. The lines should look
the same when viewed through both bottles.
4. If the culture is less turbid, incubate further.
5. If the culture is too turbid, adjust by adding sterile Mueller-Hinton Broth.

This procedure results in an inoculum with approximately 1x108 colonies/ml. Another

procedure, the stationary phase procedures are sometimes used. Several colonies are picked
and added to a bottle of Muller-hinton Broth to achieve the same turbidity as the McFarland
standard. The inoculum is then used without incubation.

Use the prepared inoculum within 20 minutes after standardization.

(C)Bauer-Kirby disk diffusion assay method

This is a test which uses antibiotic-impregnated paper disk to test whether a particular
bacteria are susceptible to specific antibiotic. A known quantity of bacteria is grown on agar
plate in the presence of a thin filter paper disc containing relevant antibiotics.

1. Dip a sterile cotton swab into the inoculum. Squeeze the cotton swab against the inner
wall of the bottle to remove excess fluid.
2. Spread the cotton swab on the Meller Hinton agar plate and swab evenly.
3. Repeat twice, turning the plate 60 ̊ at each swab (see diagram).
 4. Replace the lid of the petri dish and hold at room temperature for at least 3 minutes to
allow surface moisture to be absorbed.
5. In about 15 minutes, place the antibiotic discs onto the surface and press firmly using a
pair of forceps. Arrange the discs so that their centers are at least 24 mm apart from
each other and 10 mm away from the edge of the agar. Alternatively, an automatic
disc dispenser can be used.
6. Within 15 minutes, invert the plates and place in incubator 37 ̊ C. At least for 16 hours.

(D)Reading results

1. Examine plates after 16 to 18 hours. A confluent lawn of growth should be observed.

If only isolated colonies can be seen, the inoculum was too light and test should be
repeated.
2. Measure the diameter of the zone of inhibition for each antibiotic (including the
diameter of the disc). The end point should be the area showing no distinct growth as
judged by the unaided eye.
3. Using the information provided by the manufacturer of the discs, interpret the results
as resistant, intermediate, or susceptible.

(E)The MIC assay

1. Prepare 256 ug/ml solution of several types of antibiotics.

2. Filter sterilize each antibiotic solution.
3. Pour about 15 ml of sterile Mueller Hinton broth into a sterile petri dish.
4. Using a micropipettor, take 50 ul of the broth into each well of 96-well microtiter
plate.
 5. Add 50 ul of antibiotic solution to the first well of the first rows.
6. Repeat with other solutions.
7. Serially dilute the antibiotics in the first column by mixing and then transferring 50 ul
to the next column if wells.
8. Repeat until the eleventh well. (What is the concentration in the eleventh well?)
9. Do not add antibiotic into the last column of wells. Add 50 ul of Mueller-Hinton broth
instead. This will serve as control.
10. Dilute the standardised bacteria inoculum 1:100 (how?)
11. Add 50 ul of this to each row of well. (Calculate no. of cfu added per well).
12. Incubate at 35 ̊ C for 16 to 20 hours and read the results.

Observations:

Disk assay: Collect all data from your group and fill in the table below:

Inhibiton zone diameter (mm)

Disc Antimicrobial agent Content(ug) Resistant Intermediate Susceptible

 Inhibition Zone diameter (mm)

Disc S.aureus B.subtilis E.coli P.aeruginosa

EXPERIMENT 2: MICROFLORA OF THE SKIN

Objectives: After this exercise, the student should be able to:

A. Perform a skin swab

B. Presumptively identify Staphylococcus aureus in pus sample
C. Perform the catalase and coagulase test to identify S.aureus
D. Prepare blood agar

Introduction:

The skin supports a large number of microorganisms which normally grow without
harming the host. This is known as the normal flora. In fact, the presence of the normal flora
helps protects the skin from infection by competitions of space and nutrients. Some of these
organisms, however, can attack the human body in immunocompromised hosts.

An important pathogen that is always found on the skin Staphylococcus aureus. It will
cause an infection in the events when the immune system is weakened, when there is injury
and when a medical device is inserted. S. aureus and another staphylococci, s. epidermis, are
major causes of nosocomial or hospital-acquired infections (infections acquired during a stay
in the hospital). The emergence of Methicilin-resistance S.aureus, or MRSA, is a major
medical concern since this bacteria is resistant to almost all available antibiotics.

The major groups of organism found in skin and wound infection include gram
positive cocci, Pseudomonas and some enterobactericeae. Clostridium may infect deep
anaerobic wounds. Blood agar is usually employed as a general culture medium while
MacConkey Agar is used to recover Gram negative bacteria. If S.aureus is suspected, a
Mannitol Salt Agar plate is also inoculated.

In this practical, we will perform a skin swab and see the diversity of organisms that is
present. We will also culture a pus sample. We will perform some tests for the identification
of S.aureus. You will also learn to prepare blood agar.

Material:

• 2 sterile cotton swabs

• 2 tubes of 100ml peptone water
• 1 flask 100 ml blood base; molten at 55˚C
• 1 bottle 5 ml human blood
• 6 empty petri dishes
• 4 Mannitol Salt Agar plates (MSA)
• 2 MacConkey agar plate
• 1 bottle 3% H2O2 (Hydrogen Peroxide)
• 1 bottle 5% Calcium chloride
• 2 sterile test tubes
• 2 glass slide
 • 1 24 hour plate culture of S.aureus on brain heart infusion agar
• 1 24 hour plate culture of S. pyogenes on brain heart infusion agar
• 1 bottle of pus swab sample in Stuart’s transport medium

Procedures

(A) Preparation of blood agar

1. Remove the 100 ml molten blood agar base from the water bath and cool to touch.
2. Aseptically add 5 ml of the human blood
3. Swirl to mix well, but avoid bubbles
4. Pour into 6 agar plates and allow to solidify.

(B) Skin swabs

1. Using a sterile swab, swab the forearm of your partner.

2. Place the swab into peptone water and mix.
3. Pick up one loopful and streak on a Blood agar plate.
4. Also, streak one loopful and streak on a MacConkey agar plate.
5. Incubate at 37˚C for at least 18 hours.

(C) Pus sample

1. Streak one loopful of the pus sample onto a blood agar plate.
2. Streak one loopful onto an MSA plate.
3. Incubate at 37˚C for at least 18 hours.

(D) Test for S.aureus

A gram stain showing Gram positive cocci will indicate the presence of Staphylocci or
streptococci.

a.To differentiate between Staphylococci and streptococci:

1. Streak half (1/2) of a blood agar with S.aureus. Streak the other half with streptococci
pyogenes. Incubate 37̊C for at 24 hours.

b. Perform the catalase test

2. Transfer some cells from the middle of S.aureus colony onto a glass slide.
3. Add 3 drops of 3% H2O2. Watch for instant and sustained release of bubbles.
4. Repeats with S. pyogenes.

c. To differentiate between the two most common Staphylococci. S.aureus and S.epdemidis.
Test for fermentation of mannitol-(S.aureus +ve ; S.epidermidis –ve)

5. Streak half (1/2) of MSA plate with S.aureus. Streak the other half with S.epidemidis.
6. Incubate at 37̊C for 24 hours.

d. Perform the slide coagulase test -(S.aureus +ve; S.epidemidis –ve )

1. Add 0.5 ml of plasma into a sterile test tube.

2. Add 0.5 ml of S.aureus broth culture.
3. Mix well and incubate at 37 ̊ C.
4. Observe every 30 minutes for clot formation.
5. If no clot is formed by 4 hours, continue to incubate and observe again after 18 hours.

Note: If a gram stain indicates Gram positive cocci, Staphylococcus sp. and streptococcus sp.
should be suspected. A catalase test should then be carried out. There are three common
species which are common in infections: S.aureus, S.epdemidis and S.saprophyticus can be
identified by its resistance to the antibiotic novobiocin.

We will study S.pyogenes in the next practical.

Checklist: Each pair should have at the end of the practical

1 Blood agar - skin swab

1 MacConkey agar -skin swab

1 Blood agar -pus sample

1 MSA -pus sample

1 Blood agar -staph & strep

1 MSA -two types of staph

1 Tube of coagulase

Observations:

(B) Skin swabs on BA. Count how many different types of colonies formed. Describe
the morphology of the predominant colonies.

Skin swabs on MSA. Describe as instructed above.

 (C) Pus sample on BA and MSA. Describe as instructed above.

(D) Tests for S.aureus

S.aureus S. pyogenes
Hemolysis on BA

Catalase

S.aureus S. epidermidis
Slide coagulase test

Tube coagulase test

Discussion:

Conclusion:
Question:

1. The blood agar has to be sterile. How do you sterilize blood?

2. For catalase test, it is not recommended to use cultures from a blood agar plate. Why?

3. In the catalase test, a slow reaction with a few bubbles is not considered positive.
Why?

4. In this practical, what are the functions of the blood agar, MacConkey agar and MSA?

5. You received a pus sample and was asked to check if its contain S.aureus. How will
you do it to obtain results in the shortest time? Draw your flowchart on the blank page
opposites.
EXPERIMENT 3: URINARY TRACT INFECTION

Objectives: After this exercise, the student should be able to :

(A) Perform a macroscopic and microscopic examination.

(B) Perform and semi-quantitative culture of a urine sample.
(C) Identify the presence of Enterobacteriacea and Pseudomonas in urine.
(D) Perform tests to differentiate major groups of the Enterobacteriacea.

Introduction:

Freshly produced urine is sterile, but as it travels down the urethra, it can pick up
contaminants. The presence of bacteria in urine, thus may or may not indicate an infection.
Urinary tract infection can is usually due to organisms coming in from outside and entering
the urethra, and is more common in female.

The most common organisms encountered are members of the Enterbocteriacea,

especially E.coli. Other etiological agents include gram positive cocci, Pseudomonas
aeruginosa, anaerobic bacteria, chlamydia and mycoplsamas. Urine samples are usually
cultured on MacConkey Agar to recover Enterobacteriacea and blood agar to recover gram
positive cocci. If other organisms are suspected, selective media can be employed.

Material :

• 4 calibrated loops
• Triple Sugar Iron slants
• SIM medium
• Voges-Proskauer medium
• Simmon’s Citrate medium
• Urea test broth
• MacConkey agar plates
• Eosin Methylene Blue Agar
• 2 urine samples from student- A&B
• Glass slide
• Microscope
• Plastic Conical Tube
• Appendorf tube

Either

1. 1 24 hr plate culture of E.coli on MacConkey Agar

2. 1 24 hr plate culture of Citrobacter sp on MacConkey Agar

Or

3. 1 24 hr plate culture of Serratia marcescens on MacConkey Agar

4. 1 24 hr plate culture of Proteus vulgaris on MacConkey Agar
(A) Macroscopic examination

1. Observe the urine sample for the transparency, noting its quantity, color, clarity or
cloudiness, odor and etc.

(B) Microscopic examination

1. Place about 10mL in plastic conical tube labeled with patient's name and cover tube with
tight fitting cover.

2. Place tube in a centrifuge and place a second tube containing an equal amount of water
(with tight fitting cover on) directly opposite tube with urine, to act as counterweight.

3. Centrifuge urine specimen according to manufacturer's directions at an RCF (relative

centrifugal force) of 400 x g for 5-10 minutes.

4. After centrifuge has stopped, remove the tube and pour off the supernatant, leaving any
sediment in the bottom of the tube.

5. With a plastic pipette, mix the remaining liquid and sediment and remove a few drops of
the mixture. If there is no obvious sediment present, remove a few drops of urine from bottom
of tube.

6. Place one drop of the sediment solution on a glass slide and cover with a cover slip.

7. Using the microscope, examine the sediment using phase contrast or bright light under low
(10x) and high (40x) power, scanning several fields to obtain average numbers of formed
elements.

(C) Semi-quantitative culture of urine samples.

1. Open the Sample A bottle aseptically.

2. Take a loopful of the sample A.

3. Streak a MacConkey agar plate using the pattern show below:

First streak a straight line down the middle of the agar. Without flaming the loop or taking
any more samples, streak across the line in a zig-zag pattern until the entire plate is
covered.

4.Repeat the steps using sample B.

5.Incubate at 37 ̊ C for 24 hours.

On the next day:

6. Count the colonies on each plate and multiply by 1000. Report as the number of
colonies/ml.

Interpret the results as follow:

< 10,000 - not significant

10,000 to 100,100 - borderline, repeat the test

>100,000 -significant, identify and do sensitivity tests.

7. From the MacConkey agar plate inoculated with sample A, pick a representative lactose-
fermentor colony and streak onto half of Eosin Methylene Blue Agar plate.

8. Perform an oxidase test.

9. Also inoculate into one tube of Triple Sugar Iron.

10. From the MacConkey agar plate inoculated with sample B, pick a representative non-
lactose fermenter colony and repeat steps 7-9.

11. Incubate at 37̊ C for 24 hours.

(D) Biochemical tests for the differentiation of Enterobacteriaceae.

Various bacteria from the enterobactericeae family are frequently isolated from urinary tract
infection. Although in most cases complete identification is not necessary, it is important to be
able to identify major groups. Nowadays, this is most conveniently achieved by using rapid
kits such as the API system. Nevertheless, the ability to perform an identification scheme is
still required for a microbiologist worth his salt.
You will be provided with 4 cultures on MacConkey Agar. Work in groups of eight; four
pairs. Each pair will run six tests on each bacteria. At the end, compile your results and fill in
the identification table in the Observation section.

1. Note down the colony morphology of each organism on MacConkey agar.

2. Inoculate each organism into SIM medium, Voges-Proskaueur medium, Simmon’s
Citrate medium, KCN medium and urea test broth.
3. Perform the ONPG test. Suspend one loopful of bacteria in 0.5 ml physiological saline
in an eppendorf tube. Add one drop of toulene and vortex and shake vigorously for a
few seconds. Add 0.5 ml of the ONPG test solution.
4. Incubate at 37 ̊ C.

On the next day:

5. Add 15 drops of Kovac’s reagent to the SIM medium. The development of a red color
within a few seconds indicate a positive test.
6. Transfer 1 ml of the Voges-Proskaueur medium to a clean test tube. Add 0.6 ml α-
naphtol followed by 0.2 ml of 40% KOH. Shake the tube gently and stand for 15
minutes. The formation of red color indicates a positive test. Read the results within
one hour.
7. Observe the Simmon’s citrate medium. A blue color indicates a positive reaction. If no
blue color is present, check for the growth of the colonies. Presence of colonies also
indicates a positive reaction,. In this case, further incubate another 24 hours and check
again.
8. Check the KCN medium. A turbid medium indicates positive growth.
9. Check the urea broth. Red color indicates a positive reaction.
10. Check the ONPG test. Yellow color indicates a positive reaction.
Checklist: Each pair should have at the end of the practical

1 MacConkey Agar -Sample A

1 MacConkey Agar -Sample B

6 Biochemical tests

1 EMBA -Sample A&B

2 TSI -Sample A&B

Oxidase test -Sample A&B

Observations:

(A)Semi-quantitative urine culture

Sample A =____________________ colonies/ml

Sample B=_____________________ colonies/ml

Sample A Sample B
TSI reaction

Oxidase Test

Colony on EMBA

Preliminarily identified as:

(B) Biochemical tests for differentiation of Enterobacteriaceae

Colony morphology on MacConkey :

E.coli

Citrobacter sp.

Serratia marcescens

Proteus vulgaris
Test Escherichia Citrobacter Salmonella Klebsiella, Yersinia Proteus,
coli Enterobacter, Morganella,
Serratia Providencia

H2S (in TSI)

Indole
Voges-proskaueur
Citrate
KCN (growth in)
ONPG
Phenylalanine
Deaminase
Urease

Lysine
decarboxylase

Discussion:

Conclusion:
Question:

1. Urine samples are always collected as ‘mid-stream,clean catch” samples. Describe.

2. Usually, before urine is cultured, a microcopic examination is conducted to look for

other indicators of infection. Give a few examples of these indicators.
Experiment 4: blood cultures

Objectives: After this exercise, the student should be able to

A. Set up blood cultures with various media.

B. Read blood cultures reactions.
C. Design a scheme to identify the infective agent.

Introduction:

Blood is normally a sterile fluid. The presence of bacteria in the blood gives rise to
bacteremia, which may develop into highly fatal septicemia. Consequently, all bacteria that
are detected in the blood are considered pathogenic. Organisms commonly found in the blood
are again part of the normal flora: the Gram +ve cocci, Gram –ve rods, Pseudomonas etc. This
organism may be present at a low level and direct isolation may not give results. Normally, a
blood culture is set up, where a certain amount of blood is collected aseptically and added to a
bottle of medium. This is then incubated for up to one week and checked daily. Upon positive
growth, a subculture is made to an agar plate for isolation and identification.

Various media may be used: Trypticase soy broth, thioglycollate broth and columbin
broth for general isolation, brain, heart infusion broth for fungi and Brucella broth and
Dextrose phosphate broth for Brucella. Usually, an anaerobic broth is also set up. It is also
recommended that a hypertonic broth is set up at the same time to recover cell-wall deficient
strains. This is achieved by using a general purpose broth, e.g. Columbia broth or Brucella
broth with added 10% sucrose.

The appearance of the culture after incubation usually provide clues regarding the type of
organism that is present (see Appendix). Subculture should be done within two days to
recover the organism. General purpose isolation medium such as MacConkey, Blood and
Sabouraud Dextrose agar inoculated. If particular bacteria are suspected, a selective agar may
be employed.
Materials:

• 1 Anaerobic Jar with Gas-Pak

• 1 set Gram stain reagent

• 1 Sabouraud dextrose agar plate

• 1 Blood agar plate

• 1 MacConkey Agar plate

• 1 bottle 50ml Thioglycollate broth

• 1 bottle 50ml Trypticase soy broth

• 1 bottle 10ml blood sample

• Various reagents and test medium

Procedure:

A) Setting up of blood cultures

1. Aseptically add 5ml of blood to the Thioglycollate broth and Trypticase soy broth.

2. Mix gently. Avoid bubbles.

3. Incubate upright at 35oC for up to 7 days. Check daily.

4. Once growth is evident, observe the characteristics of the growth.

5. Choose the culture bottle with the best growth and streak on all the type of medium
provided.

6. Incubate as appropriate and check for colonies.

7. Perform Gram stain.

8. From the data available, make presumptive identifications.

9. Conduct a few chemical tests to confirm.

The blood samples have been deliberately spiked with normal flora organisms commonly
implicated in bacteremia. Remember, this is a lab practical and it may all seem very easy. In
a real-life clinical situation, you will be faced with a huge variety of possibilities. This is why
logical and analytical thinking is important. Make sure you learn the roped from a competent
clinical microbiologist before you attempt this type of work.

Observations:

Sample number:

Blood cultures appearance (Take note of tell-tale sign like hemolysis, bubbles, changes in
color, etc. Refer to the Appendix)

Colony morphology

MacConkey Agar

Blood Agar

Sabouraud Dextrose Agar

Gram stain results

Presumptive identifications:

What other tests will you do to confirm and why? Show your flowchart on the opposite page.

Do your tests now and record the results.

Experiment 5: cerebrospinal and other body fluids

Objectives: After this exercise, the student should be able to:

A. Process a CSF specimen or other body fluid specimen

Introduction:

The cerebro-spinal fluid (CSF), which completely surrounds the brain and spinal cord,
is normally sterile fluid. Infection by microorganism usually involves the meninges, a
membrane surrounding the brain and spinal cord (meningitis). The infection is acute and life-
threatening, and thus the examination of CSF is an emergency procedure. Among the
organisms that can cause meningitis is Streptococcus pneumonia, Streptococcus agalactiae,
Haemophilus influenza, Neisseria meningitides, Gram -ve bacilli, Listeria monocytogenes.
Staphylococci, Leptospira, Treponema pallidum, Mycobacterium tuberculosis, Cryptococcus
neoformans, Candida, toxoplasma gondii, Naegleria and Acanthamoeba.

CSF specimens are collected under strict aseptic conditions to avoid contaminations
and false-positive. The specimen must be processed as soon as possible to maximize recovery
of the organism. The specimen must not be refrigerated, since some of the organisms are very
sensitive to cold. A Gram stain is usually performed to look for characteristics organism. The
sample is then centrifuged at 1,500g to concentrate the organisms in isolation. Both the
sedimented cells and the supernatant should be cultured. General purpose media such as,
Blood, Chocolate and MacConkey Agar are inoculated while selective media are used for the
more exotic organisms. The CSF specimen will also be tested for viral and other antigens
using serological methods.

Other body fluids such as aspirates, pleural fluid specimen, etc. are similarly treated, although
not as medical emergencies.
Materials: (per group of 4)

• 1 sterile 50ml centrifuge tube with anti-aerosol cap

• 1 set Gram stain reagent
• 2 Blood agar plate
• 2 MacConkey Agar plate
• 1 bottle 10ml CSF sample
• Various reagents and test medium

Procedures:

Select a sample that forms the same group as your sample in experiment 5. That is, if you
tested sample in #3 in exp 5, use sample for this practical too.

1. Perform a Gram stain.

2. Working in a biological safety cabinet, transfer the CSF specimen into a 50ml
centrifuge tube.
3. Centrifuge in the Sorvall RC5C centrifuge using a SS34 rotor at 4000rpm.
4. Recover the tube. Streak both the supernatant and the sedimented cells onto all 3 types
of media provided.
5. Incubate as appropriate and observe daily.
6. Choosing a typical colony, perform a Gram stain.
7. Make a presumptive identification.
8. Perform additional tests to confirm the identity.

Usually, a chocolate agar is also inoculated to check for H.influenzae and N.meningitidis.
Here we will omit it as these organisms will not be in the doctored sample. It is also
recommended that a thioglycollate broth be inoculated. Some labs will inoculate a
Lowenstein-jensen media to recover M.tuberculosis. The presence of protozoa, fungi or yeast
can usually be observed microscopically.
Observations:

Sample number:

Appearance of the sample

Gram stains results:

Colony morphology

MacConkey Agar

Blood Agar

Sabouraud Dextrose Agar

Gram stains results:

Presumptive identifications:

What other test will you do to confirm and why? Show your flow chart on the opposite page.

Do your test now and record the results.

Confirmed identification: