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Student Guidance for Practical Work – Microbiology— (Pharmacy)
1

 Respect yourself by wearing APPROPRIATE clothing and shoes when attending lectures and practical work. Do not wear t-shirts, jeans nor sandals.
 SILENCE any handheld/gadget during lectures and practical work.
 PREPARE these following equipments for practical work:

Color pencil
 Waterproof marker
 Napkin, antiseptic soap, tissue

READ your text book and practical work guidance and preparation carefully before entering the laboratory.
 Modules must be done BEFORE entering lectures.
 Students must BRING their practical work guidance book throughout the practical work.
 Students must arrive TEN MINUTES before lectures and practical work.
 Students are NOT allowed to eat or drink during practical work.

Wear your LABORATORY COAT before entering the laboratory and during practical work. Your NAME TAG must be on your left chest.
Malang, 15 September 2013
Head of Department of Microbiology
Faculty of Medicine, Brawijaya University
Prof. DR. dr. Noorhamdani AS., SpMK(K)
Student Guidance for Practical Work – Microbiology— (Pharmacy)
2


Remember that you are facing pathogenic bacteria.

Prepare this following equipment: o Color pencil o Waterproof marker o Match o Tweezers o Napkin, antiseptic soap, tissue

Read your text book and your practical work instruction book carefully so that you understand what you are going to do in the practical work.

Wear your lab coat/apron when you are in the lab and during practical work. Your name tag must be on your left chest.

Work carefully.

If you break any of the practical work equipment, you must replace it with the new one.

Everytime you are about to start working, you must first check the completeness of the equipment/material prepared in your desk. You should also check whether the microscope is in good condition.

If there is something you lack of, tell the instructor.

During practical work, you are not allowed to smoke, eat, or put your fingers or other things inside your mouth.

If accident happens (even small accident), for example injury or the bacterial culture is spilled in great amount, quickly tell the instructor.

If the bacteria spill onto the table, your skin, or your lab coat, quickly clean it with cotton moistened with Lysol solution or 70% alcohol.
 Put the used equipment (for example: pipette, object glass, swab, or other equipment) in lysol solution.
 Ose, tweezers, etc must be burned first before you store them.
 Bacterial culture must always be closed when not used.
 Put the waste in the waste bin/trash. Do not throw out tissue, cotton, or paper into the drainage.
 The staining process must be done in the staining rack.

Put out the Bunsen burner when not used.
Student Guidance for Practical Work – Microbiology— (Pharmacy)
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
Clean the objective lenses (100x) with lens paper or tissue.

Turn off the taps, fire, and lamps.

Clean up the bench and put back all the material/equipment into its proper place.

Wash your hand using antiseptic soap.

You are not allowed to take the bacterial culture home.

After every practical work, you must make a report according to the instructor’s instruction.

Use the empty pages of this instruction book to write down the result of your practical work.
Student Guidance for Practical Work – Microbiology— (Pharmacy)
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Arrange the table and chair so that the ocular lens of the microscope is eye-level.
Try to look using both eyes opened to prevent eyestrain.
1.
Check the cleanness of the lenses, the objective lenses and the ocular lens.
2.
Adjust the lens position with the light source.
3.
Condenser’s position:
In the microbiology laboratory, we examine very small bacteria, so we use objective lenses with 100x magnification (using the immersion oil) whereas the focal length is short (2 mm).
In order to get as powerful and as much light as possible into the objective lenses, place the condenser as high as possible. The very high position of the condenser will cause the air volume between the condenser and the object glass becomes very little, so that the entering light rays is not refracted, and the light coming into the object glass will have enough quality and quantity.
The opposite will happen if the condenser is placed in the lower part.
4.
Adjust the amount of light entering the range of visibility in order to get optimum and focused illumination by:
-
Close the condenser diaphragm.
Adjust the position of the mirror/lens so that the inside part of the diaphragm can be seen clearly. This means that the amount of light needed is enough.
Next, open the diaphragm again.
5.
Place the preparation which is going to be examined on the stage.
6.
Use low power objective lenses first to chose the area which is going to be examined, that is the thinner part of the preparation, where the bacteria are not clumped. Put into focus the preparation using these low power objective lenses by rotating the coarse-adjustment first, and then the fine-adjustment.
7.
When the area to be examined is found, change the magnification of the objective lenses into 100x magnification. Use the immersion oil.
The immersion oil is used to remove the refractive power of air between the preparation/object glass and the objective lenses, so that more light is entering the objective lenses because the refractive index of immersion oil is almost the same as the refractive index of glass.
Besides immersion oil, other kinds of oil or other things that have almost the same refractive index as glass can be used, for example: Canada balsam, xylol, balsam, eupara, and glycerol.
Student Guidance for Practical Work – Microbiology— (Pharmacy)
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8.
After you drop the immersion oil (one drop) to the object glass, rotate the coarseadjustment knob until unclear image is visible. Rotate the fine-adjustment knob to bring the image into focus.
9.
After using the microscope, clean the objective lenses using lens paper or cotton moistened with a little xylol/benzene to remove the immersion oil.
Then, clean the remaining xylol/benzene using dry lens paper.
Student Guidance for Practical Work – Microbiology— (Pharmacy)
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The examination in the Microbiology Laboratory is aimed to find out exactly the kind of bacteria which causes an infectious disease. This is essential to help the practitioner making the diagnosis. In order to get the expected result, a laboratory technician must follow certain conditions and obey the examination procedures.
After the specimen is received and recorded in log book, do the examination following the procedure below.
3.1.1 Direct smear and staining
Make the smear from the specimen at the object glass, then stain and observe under microscope. o The routine staining done is Gram staining. o Depending on the clinical diagnosis, certain staining can be carried out, for example:
Metachromatic/Neisser staining for diphtheria.
Acid fast staining for TBC and leprosy.
-
Spore staining for anthrax and tetanus.
By staining, you can identify the morphology and the characteristic of the bacteria against staining.
For certain specimens, you can make wet preparation without staining, for example when you want to see living bacteria:
Use hanging drop method to see bacteria movement
Use darkfield microscope to see Leptospira
3.1.2 Bacterial culture
For bacterial culture and primary isolation, use the appropriate culture medium for its growth, for example:
C. diphteriae : PAI, Loffler medium
M. tuberculosis
N. gonorrhoe
Streptococcus sp.
D. pneumoniae
: Lowenstein Jensen medium
: Thayer Martin V C N medium
: Blood Agar Plate medium
: Chocolate Agar Plate medium
Enterobacteriaceae : Mc Conkey, Eosin Methylene Blue medium
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After inoculation or streaking on culture medium, incubate the medium in the incubator using optimum temperature 35-37°C for 18-24 hours.
Certain bacteria need anaerobe atmosphere for growing, so the medium must be put into anaerobic jar. Some bacteria need 5-10% CO
2
for growing; therefore the medium must be put into candle jar.
If there is bacterial growth (colony) after incubation, bacterial identification can be carried out.
Bacterial identification isdetermined according to the morphology found at the direct smear examination and the form, the color/pigment of the colony at culture medium.
3.1.3 Biochemical reaction
To identify bacteria, we can do several biochemistry reactions, such as sugar fermentation test, indol production, urease production, and many other biochemical reactions.
3.1.4 Sensitivity against antibiotic/antimicrobial test
The purpose of this test is to find out whether pathogenic bacteria are sensitive towards in vitro antimicrobial. This test helps clinician in giving therapy.
3.1.5 Virulence/pathogenicity test
The purpose of this test is to find out whether the pathogenic bacteria are pathogen/toxigenic or not.
This test can be carried out:
In vitro, for example coagulase test for Staphylococcus aureus.
In vivo, using experimental animals (such as: rabbit, mouse, guinea pig) infected by bacteria.
Selecting the experimental animals is based on its sensitivity towards the examined bacteria.
Another thing to be considered when carrying out microbiological examination is that every transportation or collection of bacteria from one medium to another must be in aseptic condition in order to prevent contamination.
Student Guidance for Practical Work – Microbiology— (Pharmacy)
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4.1 Staining
In this practical work you will learn how to do several staining technique which are important for clinical needs.
Making the smear for staining

Clean the object glass with cotton or tissue and pass it above fire to remove grease. Allow it to cool.

Make one or two circles with diameter 2-3 cm on one side of object glass. Write label to each circle if necessary.

Flip the object glass and clean the surface from fingerprints and other debris by using tissue.
Pass the object glass over bunsen once to three times.

Prepare the smear. Make it not too thick and not too thin by: o Smear from solid culture
Drop one ose of sterile aquadest onto the object glass. Take some bacterial culture using an ose, and then suspense it with the aquadest on the object glass and spread evenly. o Smear from broth culture
Drop one ose of the broth culture onto the object glass and spread evenly. Since broth media is liquid, therefore sterile aquadest is not needed in making the smear.

Allow the smear to air dry, then heat-fix it on the Bunsen burner for three times.
4.1.1 Simple Stain
Provided:
 Bacterial culture
 Methylene blue or safranine
 Object glass and ose
 Bunsen burner
 Sterile aquadest
 Water and bibulous paper
Procedure:
1.
Make the smear on the object glass.
2.
Flood it with methylene blue or safranine for ½-1 minute.
3.
Wash off the stain with tap water and dry with bibulous paper.
4.
Observe it under the microscope using 100x magnification objective lenses. Use immersion oil.
Student Guidance for Practical Work – Microbiology— (Pharmacy)
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4.1.2 Gram Stain
Provided:
1.
Bacterial culture
2.
Stain for Gram stain
3.
Object glass and ose
4.
Bunsen burner
5.
Sterile aquadest
6.
Water and bibulous paper
Procedure:
1.
Make the smear on the object glass.
2.
Flood it with crystal violet for 1 minute. Wash off the crystal violet with tap water.
3.
Flood it with lugol for 1 minute. Wash off the lugol with tap water.
4.
Flood it with 96% alcohol for 5-10 seconds or until the stain is decolorized. Wash off the alcohol with tap water.
5.
Flood with safranine for ½ minute. Wash off the safranine with tap water.
6.
Dry with bibulous paper.
7.
Observe it under the microscope using 100x magnification objective lenses.
4.1.3
Spore Stain
Provided:
1.
Spore forming bacteria on nutrient agar slant culture
2.
Malachite green and safranine
3.
Object glass and ose
4.
Bunsen burner
5.
Sterile aquadest
6.
Water and bubilous paper
Procedure:
1.
Make the smear on the object glass.
2.
Flood with malachite green and heat for 5 minutes, do not let it boil and dry.
Wash off the malachite green with tap water.
3.
Flood with safranine for ½ minutes.
Wash off the safranine with tap water.
4.
Allow it to dry and then observe under microscope.
For spore staining, modification of acid fast staining method can also be used without using acid alcohol.
Usually one bacterium form one colony (consists of a group of bacterial cells). The morphology of these colonies sometime can help the bacterial identification, because it is specific for each bacterial species.
To study the bacterial colonies, observe:
1.
Size of the colonies
2.
Surface: is it M (mucoid), R (rough), or S (smooth); is it convex or plan, is there any pigment production?
3.
Margin of the colonies: rata, or fimbriated, or ciliated
Student Guidance for Practical Work – Microbiology— (Pharmacy)
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4.
Swarming of the colonies (specific for Proteus spp. grow in non-inhibitory media such as blood agar)
5.
Consistency of the colonies, use öse to check it
6.
Special feature, is the colonies glossy, matt; (e.g. E. coli will give metallic sheen when grown in Eosin Methylene Blue (EMB) agar)
7.
Odor of the colonies (fishy odor in Proteus spp., etc)
8.
In blood-containing media such as blood agar plate, we can watch several types of hemolysis:
α hemolysis (partial hemolysis): greenish zone surround the colonies
β hemolysis (total hemolysis): clear zone surround the colonies
-
γ hemolysis: non hemolysis
Demonstration:
1.
Escherichia coli in EMB agar plate.
2.
Proteus sp. in blood agar plate.
3.
Bacillus sp. in nutrient agar plate.
4.
Staphylococcus aureus, S. citreus, S. albus in nutrient agar plate.
5.
Peudomonas sp. in nutrient agar plate.
6.
Streptococcus spp. in blood agar plate showing several types of hemolysis.
The aim of this practical works is to get one isolated colony, pure culture which not mixed with other bacteria. This will help bacterial identification by examining:
characteristics of the colony
morphology and staining
biochemical properties
serologic reaction
antimicrobial sensitivity test
Provided:
mix bacterial culture of S. aureus and E. coli in broth
nutrient agar plates (NAP)
Gram stain
Procedure:
1.
Using waterproof OHP marker, mark your NAP into 4 parts.
2.
Take 1 loopfull of bacterial culture and streak it in the 1st part of your NAP.
3.
Red heat your ose, and let it cool.
4.
Using the same ose make another streak in your 2 nd part of your agar
5.
Do the same until you reach the 4 th part of your agar and streak it zigzag
6.
Incubate the plates at 37 0 C for 18-24 hours.
7.
Examine your plate at the following day. If you do it properly you will get isolated colonies of each species of bacteria in your 4 th part of agar.
8.
Do Gram staining for each different colony.
Student Guidance for Practical Work – Microbiology— (Pharmacy)
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In vitro test to get the pattern of bacterial sensitivity against antimicrobial drugs is urgently needed for the clinician to choose the appropriate antimicrobial drugs, because some bacteria are already resistant to the drugs.
There are two types of antimicrobial sensitivity test:
A.
Disc diffusion method
B.
Tube dilution method
4.5.1
Disc diffusion method
In this practical works we use Kirby-Bauer method using table made by CLSI (Clinical and Laboratory
Standards Institute). In this method we use small paper disc Ø 6 mm which contains antimicrobial drugs in known concentration.
Provided:
Solid media (DST – Diagnostic Sensitivity Test Agar) or Muller Hinton Agar in plates
Bacteria in broth with degree of turbidity Mc Farland 0,5 (cell density 1,5x10
Antimicrobial discs
8 bacteria/ml)
Sterile swabs
Ruler and CLSI table
Procedure:
Using sterile swabs take some bacterial inoculum from the broth
Press swabs on the tubes so there was no fluid excess, then make streaking across the agar plate, make sure that you streak evenly
Let it dry (about 2 minutes) then put the discs with this condition:
1) never take again the discs that already been attached to the agar,
2) pay attention to the distance between discs
Incubate at 37 0 C for 18 -24 hours
To determine whether the bacteria are sensitive or resistant against a specific antimicrobial drug, measure the diameter of the inhibition zone (in milimetre) and compare it with CLSI table.
Besides diffusion method (Kirby Bauer), there is another diffusion method by Joan-Stokes. In this method, we compare the inhibition zone of test microbes with known sensitive and resistant microbes (control microbes).
The criteria are as follows:
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
Sensitive: if the radius of the inhibition zone of the test microbes are the same or smaller but not > 3 mm than control

Intermediate: if the radius of the inhibition zone of the test microbes are >3 mm, but comparing the control are < 3 mm

Resistant: if the radius of the inhibition zone of the test microbes are ≤ 3 mm
Performans Standard for Antimicrobial Susceptibility Testing : Appendix 1
4.5.2
Tube dilution method
Dilution method can be done by using either broth in tubes (tube dilution method), and using solid media in petri dishes (agar dilution method).
In both methods we use antimicrobial solution in decreasing concentration by serial dilution. In agar dilution method these solution are then mixed with melted agar, let it solidify, and inoculate the agar with test microbes.
In tube dilution method, the solutions are then mixed with broth containing test microbes (bacterial density: 10 5 – 10 6 bacteria/ml).
By this method we will obtain:

MIC (minimum inhibitory concentration): the lowest antimicrobial concentration that inhibit bacterial growth

MBC (minimum bactericidal concentration): the lowest antimicrobial concentration that kill the bacteria
Provided:
6 sterile tubes
antimicrobial drug
bacteria in broth (density 10 5 – 10 6 bacteria/ml)
Muller Hinton agar
diluent (broth or normal saline)
Procedure:
1.
Mark the sterile tubes (1 – 6)
2.
Make antimicrobial solution – certain concentration (e.g. 64 μg/ml)
3.
Put 1 ml diluent into tubes number 2 – 6
4.
Put 1 ml of antimicrobial solution into tubes 1 and 2
5.
Mix thoroughly the mixture in tube no. 2 and transfer 1 ml into tube no. 3
6.
Do the same till tube no. 6, so we will get antimicrobial concentration serially diluted (64
μg/ml, 32 μg/ml, 16 μg/ml, 8 μg/ml, 4 μg/ml, and 2 μg/ml)
7.
Then put 1 ml of bacterial broth culture into each tubes
8.
Mix the mixture thoroughly, and so the final antimicrobial concentration will be 32 μg/ml,
16 μg/ml, 8 μg/ml, 4 μg/ml, 2 μg/ml and 1 μg/ml
9.
Incubate the tubes at 37 0 C for 18 – 24 hours
10.
Examine the tubes for the turbidity as the indicator of bacterial growth. The lowest concentration that shows no turbidity is the MIC.
Student Guidance for Practical Work – Microbiology— (Pharmacy)
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11.
For the MBC: inoculate Muller Hinton agar with the tubes content which show no turbidity.
Incubate at 37 0 C for 18 – 24 hours. Plate that shows no growth is the minimum bactericidal concentration (MBC).
Student Guidance for Practical Work – Microbiology— (Pharmacy)
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4.6.1
Skin antisepsis
Skin contain normal flora. With disinfection/antisepsis procedure the sum of this flora can be reduced.
Provided:
Nutrient Agar plate
Cotton
Disinfectants/antiseptic agents: soap, alcohol 70%, povidone-iodine
Procedure:
1.
Using waterproof OHP marker, mark the plate into 5 parts
2.
Put your fingerprint (without disinfectants) into one part of your NAP
3.
Then wash each of your fingers with soap, 70% alcohol, povidone-iodine, and povidoneiodine followed by 70% alcohol.
4.
Put each of those fingers into each part of your NAP.
5.
Incubate at 37 0 C for 18-24 hours
6.
Examine your plate, and compare the formed colonies in each part of your NAP
4.6.2
Disinfection of glassware
Provided:
5 pieces of glass
Disinfectants: 70% alcohol, 5% Lysol
Nutrient broth in tubes
Bacterial broth culture
Sterile water
Procedure:
7.
Put the pieces of glass into the bacterial culture
8.
Take two pieces of glass and put them into the disinfectants (each into alcohol and Lysol) for
5 minutes, flush with sterile water, then put into broth in tubes
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9.
Take two pieces of glass and put them into the disinfectants (each into alcohol and Lysol) for
10 minutes, flush with sterile water, then put into broth in tubes
10.
Use one piece left for control (without disinfectants)
11.
Incubate the tubes at 37 0 C for 18-24 hours
12.
Compare the turbidity of each tube (that shows growth of bacteria)
Student Guidance for Practical Work – Microbiology— (Pharmacy)
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|Chapter 5|
Provided:
 Staphylococcus from a patient on Nutrient Agar slant
 Empty medium: NAP and nutrient broth
 H
2
O
2
3% solution

Gram stain
 Plasma
Procedure:
Day 1: a) Take bacteria from slant and inoculate into: o NAP to get isolated colony o Nutrient broth for catalase and coagulase test
Incubate at 37 0 C overnight (both) b) Make smear from the colony on slant and do Gram staining; examine under microscope
Day 2:
1.
Take the culture you have made the day before, and observe any pigment production by the bacteria on NAP
2.
Do catalase and coagulase test using the broth culture
Procedure of coagulase test: a) Slide coagulase test
 Take a clean object glass

Make bacterial suspension on the object glass

Add 1 drop of plasma, mix it with the bacterial suspension

Result (+)  if there clumping formation. If no clumping formation  confirm with tube coagulase test b) Tube coagulase test

Take a sterile test tube

Put 0,5 ml of plasma, add 0,1 ml broth culture

Incubate at 37 0 C, and observe every 30 minutes for agglutination. If there is no reaction, incubate overnight
Procedure of catalase test:
1.
Take the remaining broth culture, and put 1drop of H
2
O
2
solution
2.
(+): if there is bubbles formation
Student Guidance for Practical Work – Microbiology— (Pharmacy)
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Demo:
1.
Slide of Staphylococcus sp. (Gram stain)
2.
Culture of S. aureus, S. albus, and S. citreus on NAP
3.
Catalase and coagulase test (slide and tube coagulase)
Provided:
 Streptococcus culture on NA slant

Blood agar plate (BAP)

Chocolate agar plate (CAP)

Gram stain

H
2
O
2
3%

Brain heart infusion (BHI) broth
Procedure:
Day 1:
1.
Take the bacterial culture from NA slant and inoculate into: a.
BAP and CAP to get isolated colony and hemolytic characteristic of the bacteria b.
BHI broth c.
Incubate BAP, CAP (in candle jar) and BHI broth
2.
Make smear from NA slant and do Gram staining
Day 2:
1.
Take the culture from incubator and observe the hemolytic type on BAP
2.
Make smears from BAP/CAP and BHI broth, do Gram staining and study the arrangement of the bacteria
3.
Do catalase test using broth culture
Demo:
1.
Gram stained Streptococcus slide
2.
Culture of Streptococcus pyogenes, viridans group, and S. fecalis on BAP
3.
Culture of Streptococcus viridans group on chocolate agar plate (CAP)
4.
Culture of Streptococcus pneumoniae on CAP
5.
Culture of Streptococcus fecalis on McConkey agar
6.
Catalase test, and bacitracin test, optochin test
5.3.1 Bacillus sp. (aerobic bacteria)
Provided:
Culture of Bacillus sp on nutrient agar slant
Student Guidance for Practical Work – Microbiology— (Pharmacy)
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Nutrient agar plate
Gram stain
Spore stain (Schaeffer Fulton method)
Procedure:
1.
Make two smears, then do Gram stain and spore stain
2.
Examine under the microscope, and pay attention to the bacterial spores (size, shape, and location – central, sub terminal or terminal)
3.
Inoculate NAP, taking the bacteria from NA slant.
Demo:
1.
Culture of Bacillus sp. on NAP and BAP
2.
Slides of Bacillus sp. stained with Gram and spore stain.
3.
Antimicrobial sensitivity test.
5.3.2 Clostridium sp. (anaerobic bacteria)
Provided:
Nutrient broth
Thioglycollate broth
Cooked meat medium
Blood agar plate
Anaerobic jar/Gaspak system
Gram stain and spore stain
Procedure:
Day 1:
1.
Do heat sock method as follows:
 Put some soil into nutrient broth
 Put the tubes in a waterbath (t: 80 0 C) for 15 minutes
2.
Take the fluid part of the nutrient broth and inoculate it into thioglycollate broth, cooked meat medium and BAP by streaking to get isolated colonies.
3.
Incubate overnight all the medium in 37 0 C; put BAP into anaerobic jar.
Day 2:
1.
Make smears from all of the medium and do Gram stain and spore stain. Study the characteristics of the spores (size, shape, location,etc)
2.
Observe any growth on the medium, and check whether there is target hemolysis found.
Demo:
1.
Slide of Clostridium tetani (Gram stain and spore stain)
2.
Stormy fermentation (Clostridium perfringens in litmus milk medium)
Provided:
Selenite/tetrathionate broth
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McConkey agar, SS agar, EMB agar
TSI agar
IMViC reaction (Indole, Methyl red, VP, Citrate)
-
Motility, urease test
Semisolid agar
Ose (loop and straight)
Task: Identification of Enterobacteriaceae
Note:

TSI agar slant
This medium contains three kinds of sugar: glucose, lactose, sucrose, and phenol red as indicator to indicate fermentation; and FeSO
4
to demonstrate hydrogen sulfide production (indicated by blackening in the butt). Glucose concentration is
1/10 from the lactose and sucrose in order that the fermentation of glucose alone may be detected. The small amount of acid production by glucose fermentation is oxidized rapidly in the slant, which will remain to alkaline pH.

Test for indole.
Inoculate tryptophan broth. Test after 48 hrs incubation by the addition of
Kovacs or Ehrlich reagent. A red color indicates production of indole from the amino acid.

Methyl red test.
Inoculate MR-VP medium, test after 48-96 hrs by adding 5 drops of methyl red indicator. A red color is positive, whereas a yellow color is read as negative.
 Voges-Proskauer test.
Inoculate MR-VP medium and test for the production of acetylmethylcarbinol after 48 hrs by adding to 1 ml of culture 15 drops of 15% α-naphthol in absolute ethyl alcohol and 10 drops of 40% KOH. A positive test is the development of red color in 15-30 minutes.
 Citrate test.
Inoculate Simmons’ citrate with light inoculum. A positive test is indicated by the production of blue color in the medium, showing that the microbes can use citrate as a sole source of carbon.

Urease test.
A heavy inoculum is spread over the surface of urea agar slant or properly emulsified in urea broth. Urease activity is observed by production of red color due to the production of ammonia. A rapid (2-4 hrs) positive test indicates
Proteus sp.

Motility test.
See motility test in chapter 4.4.

Oxidase test.
A positive reaction recognized by dark purple color in 5-10 seconds. For
Enterobacteriaceae: negative reaction. There are two types of oxidase test: o Plate method: put 1 drop of oxidase reagent (tetramethyl paraphenylenediamine dihydrochhloride solution) to the suspected colonies o Filter paper method: saturate a piece of filter paper with the reagent and make a streak taken from suspected colonies to the paper
Student Guidance for Practical Work – Microbiology— (Pharmacy)
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Identification of Enterobacteriaceae  using Enterobacteriaceae scheme
Demo:
1.
All the member of pathogenic Enterobacteriaceae (culture and biochemical reactions)
2.
Pseudomonas aeruginosa (culture and biochemical reactions)
Enterobacteriaceae scheme : Appendix 2
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|Chapter 6|
Bacteriological examination of water plays an important role in health, because:
 water is easily contaminated by sewage or other excreted matters,
 as vehicle, it may cause outbreaks of intestinal infections, such as typhoid fever, cholera, dysentery, etc.
The indicators of water pollution are:
• Coliform bacilli  Escherichia coli
• Streptococcus fecalis
• Clostridium welchii
These microbes are not pathogens, but they indicate that fecal material has entered the supply, and that the water is easily contaminated with intestinal pathogen microbes.
Coliform bacilli are the most reliable indicators of fecal pollution, because they are:
• Normal flora of GI tract.
• Easily identified (lactose fermentation  acid + gas).
The routine tests generally used are:
1.
Qualitative method: a.
Presumptive test b.
Confirmed test c.
Completed test
2.
Quantitative method: a.
Plate count method b.
Most Probable Number (MPN) method c.
Membrane filter method
6.2.1 Qualitative test
1. Presumptive test
• Only detect whether the water had been contaminated by sewage/fecal pollution
• Using: lactose broth in fermentation tubes (Durham or Eyckman tubes), gas formation: (+) after incubation for 24 - 48 hours at 35°C
• An estimation of the number of coliform bacilli is made by adding various quantities of water specimen (from 0,1-10/50 ml) to lactose broth in series of tubes  MPN method.
2. Confirmed method
• Consists of the inoculation of a specified differential selective medium such as EMB agar; culture is taken from (+) presumptive test.
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• Typical colonies followed by Gram staining; Gram negative rods found  confirmed test (+).
3. Completed test
• Pick one or more typical colonies from EMB agar - taken from (+) confirmed test - and transfer it to lactose fermentation tube and an agar slant.
• If lactose is fermented and the smear from slant culture shows gram-negative rods  completed test (+).
6.2.2
Quantitative test
1.
Plate count method
 Make serial dilution - ten fold - of water sample.
 Take 0,1 ml of each dilution and put it on a petri dish, pour melted nutrient agar, mix it and let it solidify.
 Incubate 37°C for 18-24 hours.

Count the colonies; use only petri dish with colony count 30-300 colonies/petri dish for reporting the result after multiplying it with the dilution.
• Differential coliform method
Basically it is the same with the plate count method; using selective differential medium such as EMB/McConkey Agar.
2. Most Probable Number (MPN) method
 using presumptive test in a serial 3 or 5 tubes for 3 different volume of samples (10 ml, 1 ml and 0,1 ml), a total of 9 or 15 tubes.
 in each series, the total of (+) tubes is counted  then use McCrady table for estimating the total number of microorganisms found in water sample.
McCrady table : Appendix 3
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|Chapter 7|
Objective: to study the pathogenic fungi
Provided:
Candida albicans culture in Sabouraud dextrose agar (SDA)
Filamentous fungi in SDA
Dyes from Gram staining
Lactophenol cotton blue (LPCB)
10% KOH/NaOH solution
Specimen: skin/nail scraping, and hair
Procedure:
A.
Direct microsopic examination
Put the specimen on object glass
Put 1 drop of KOH/NaOH on the specimen and cover in using cover glass
Heat carefully using Bunsen burner
Examine under microscope (objective lens 10 – 40x)
Observe yeast/budding cells, or hyphae/mycelia
B.
Staining using LPCB
Take some culture from filamentous fungi and put on a slide
Put 1 drop of LPCB and cover with cover glass
Examine under microscope (objective lens 40x)
Observe spores, hyphae (septate or non septate)
C.
Germ tube test
Take Candida albicans culture, and mix it with mammalian serum in a tube
Incubate at 370C for ca. 4 hours
Put 1 drop of this mixture on object glass, and cover it.
Examine under microscope (objective lens 40x)
Observe yeast cells with germ tube  germ tube test (+) – specific for Candida albicans.
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Appendix 1 :
Adapted from CLSI (Clinical and Laboratory Standard Laboratory Institute) Document M100-S21 (M02-A10)
Disc
Code
Antimicrobial of Chemotherapeutic Agent
Disk
Potency
Inhibition Zone Diameter to
Nearest Milimeter
Resistant Intermediate Sensitive
Penicillin and Derivates
AMP Ampicillin
Enterobacteriaceae
Staphylococcus spp
Streptococcus spp
AMC Amoxycillin+Clavulanic Acid
Enterobacteriaceae
Staphylococcus spp
Cephalosporin
10 µg
20/10 µg
≤13
≤28
≤13
≤19
14-16
14-17
≥17
≥29
≥24
≥18
≥20
30 µg CTX Cefotaxime
Enterobacteriaceae
P. aeruginosa, Acinetobacter spp, Staphylococcus spp
Neisseria meningitidis
Neisseria gonorrhoeae
Streptococcus spp β -hemolytic group
Streptococcus spp viridans group
FOX Cefoxitin
S. aureus
Coagulase negative staphylocci
CAZ Ceftazidime
P. aeruginosa, Acinetobacter spp, Staphylococcus spp
Enterobacteriaceae
Neisseria gonorrhoeae
CRO Ceftriaxone
Enterobacteriaceae
P. aeruginosa, Acinetobacter spp, Staphylococcus spp
Neisseria meningitidis
Neisseria gonorrhoeae
Streptococcus spp β-hemolytic group
Streptococcus spp viridans group
Aminoglycoside
CN Gentamycin
Enterobacteriaceae, P. aeruginosa, Acinetobacter spp,
Staphylococcus spp
NET Netilmicin
Enterobacteriaceae, P. aeruginosa, Staphylococcus spp
AK Amikacin
Enterobacteriaceae, P. aeruginosa, Acinetobacter spp,
Staphylococcus spp
Macrolide
E Erythromycin
Staphylococcus spp, Enterococcus spp
Streptococcus spp β-hemolytic group, viridans group, S. pneumoniae
Quinolon
CIP Ciprofloxacin
Enterobacteriaceae, P. aeruginosa, Acinetobacter spp,
Staphylococcus spp, Enterococcus spp
Neisseria meningitidis
Neisseria gonorrhoeae
LEV Levofloxacin
30 µg
30 µg
30 µg
10 µg
30 µg
30 µg
15 µg
5 µg
5 µg
≤21
≤24
≤14
≤17
≤22
≤14
≤25
≤19
≤13
≤24
≤12
≤12
≤14
≤13
≤15
≤15
≤32
≤27
15-17
18-20
25-25
15-22
26-27
20-22
14-20
25-26
13-14
13-14
15-16
14-22
15-20
16-20
33-34
28-40
≥22
≥25
≥18
≥21
≥31
≥26
≥23
≥34
≥31
≥24
≥28
≥23
≥21
≥34
≥35
≥24
≥27
≥15
≥15
≥17
≥23
≥21
≥21
≥35
≥41
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Enterobacteriaceae, P. aeruginosa, Acinetobacter spp, Enterococcus spp, Streptococcus spp β-hemolytic group, viridans group, S. pneumoniae
Staphylococcus spp
Carbapenem
MEM Meropenem
Enterobacteriaceae
P. aeruginosa, Acinetobacter spp, Enterococcus spp, Staphylococcus spp
Neisseria meningitidis
Tetracycline and Derivates
TE Tetracycline
Enterobacteriaceae, Acinetobacter spp
Enterococcus spp, Staphylococcus spp, Vibrio cholera
Neisseria gonorrhoeae
DO
Streptococcus spp β-hemolytic group, viridans group, S. pneumoniae
Doxycycline
Enterobacteriaceae
Acinetobacter spp
Others
C
Enterococcus spp, Staphylococcus spp
Chloramphenicol
Enterobacteriaceae, Enterococcus spp, Staphylococcus spp, Vibrio cholerae
Neisseria meningitidis
S. pneumoniae
F
Streptococcus spp β-hemolytic group, viridans group
Nitrofurantoin
Enterobacteriaceae, Staphylococcus spp, Enterococcus spp
SXT Trimethoprim- Sulfamethoxazole
Enterobacteriaceae, Acinetobacter spp, Staphylococcus spp,
Haemophilus spp, V. cholerae
Neisseria meningitidis
S. pneumoniae
VAN Vancomycin
Enterococcus spp
Streptococcus spp β-hemolytic group, viridans group, S. pneumoniae
10 µg
30 µg
300 µg
1.25/ 23.75 µg
30 µg
30 µg
30 µg
≤13
≤12
≤19
≤20
≤17
≤14
≤10
≤11
≤14
≤30
≤18
≤10
≤9
≤12
≤25
≤15
≤14
≤15
≤19
≤13
14-16 ≥17
≥19
≥23
≥16
≥30
≥18
≥26
≥21
≥21
≥17
≥16
≥15
≥19
≥38
≥23
≥14
≥13
≥16
≥30
≥19
≥17
≥17
© 2011
16-18
20-22
14-15
13-17
20-25
18-20
15-16
11-15
12-14
15-18
31-37
19-22
11-13
10-12
13-15
26-29
16-18
15-16
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