Medical Microbiology BIOM 322
Laboratory Manual
A Laboratory Manual
Medical Microbiology
(BIOM 322)
Pre-request Microbiology (1062 241)
Organized by: Dr. Adil Makkiya
Dr. Asma Al-Thani
Dr. Hala Bargal
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Course work details divided into weeks
Course objectives divided according to course work weeks of
Medical Microbiology (1066322)
Experiment 1: Laboratory safety, precautions and sterilization & preparing culture
media their classification dispensing
Experiment 2: Use of blood differential and Selective Media
Experiment 3: Microbial flora of throat, interteeth ,skin ,nails and hair
Experiment 4: a) Identification of Human Staphylococcal Pathogens
b) Chemical agent of control, Chemotherapeutic agents
Experiment 5: Identification of Human Streptococcal Pathogens
Experiment 6: Identification of Streptococcus pneumoniae
Experiment 7: Intestinal Pathogens in the Family Enterobacteriaceae
(Characterization of lactose fermenting enteric bacteria (E. coli &Klebsiella)
Experiment 8: Characterization of non lactose fermenting enteric bacteria
(Proteus&Pseudomonas)
Experiment 9: Characterization of Salmonella and Shigella
Experiment 10: a) API -20E
b) Demonstration
Experiment 11: Haemofilus influenzae, Neisseria ,Campylobacter &Identification of
isolated unknown bacteria
Experiment 12: Introduction to Mycology diagnosis
Experiment 13: ELISA, Coomb‫׳‬s& Haemagglutination
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Course work details divided into weeks
1. First week:
Laboratory safety & sterilization techniques & preparing routine primary isolation media
(enriched, selective, differential, nutrient) description for the purpose of each media incubate
media (Atmosphere, Temperature, Humidity, Length of incubation) dispensing methods.
2. Second week:
The use of blood as differential & selective and enriched & standard inoculation and streaking
techniques (swab, loop flaming, streaking for isolation, stab) normal microbial flora of throat &
interteeth & skin & nails and hair.
3. Third week:
Results of previous section & Differentiate colony morphologies and characterization of human
Staphylococcus pathogen (salt tolerance growth & fermentation-colonial pigementation - DNase haemolysis).
4. Fourth week:
Results of previous section (colony characteristics size, shape, elevation, form and margin,
surface appearance, changes in media) biochemical reactions (catalase, coagulase). Chemical
agents of control: Chemotherapeutic agents (novobiocin sensitivity).
5. Fifth week:
Results of previous section & Differentiate colony morphologies and characterization of human
Streptococcus & Enterococcus & Streptococcus pneumonia (Differentiate common growth
characteristics with 5% Sheep Blood agar), Bacitracin test.
6. Sixth week:
Results of previous section Differentiate hemolysis alpha beta gamma (none), Differentiate
between (Streptococcus viridans & Streptococcus pneumonia) by bile solubility, Inulin fermentation
& optochin test.
7. Seventh week:
Characterization of Enterobacteriaceae (Lactorse fermenting enteric bacteria E.coli – Klebsiella
sp) differentiate common growth characteristics on Mackonckey.
8. Eighth week:
Biochemical reactions of L.F (Indole – MR/VP – Citrate utilization) + colonial morphology.
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Characterization of non lactose fermenter (Proteus & Pseudomonas).
9. Nineth week:
Colonial morphology of Proteus & Pseudomonas. (Oxidase – Urease tests).
Usage of indicators and identification of positive and negative results of IMVC.
Characterization of Shigella & Salmonella + TSI test.
10. Tenth week:
Identification of different results of TSI test + colonial morphology of Salmonella & Shigella.
API – 20E Diagnostic system for Enterobacteriace.
Demonstration of the following items:
1. Slides: a- Mycobacterium tuberculosis.
b- Clostridium titani.
2. LoffleŕsSerum
3. L.J medium.
4. Cooked meat broth medium.
5. Anaerobic gas packs system.
11. Eleventh week:
Campylobacter, Haemophylus influenzae, Neisseria.
Scheme of identification of isolated unknown.
12. Twelvth week:
Introduction to Mycology diagnosis (classification, taxonomy & morphologic structure).
Characterization of Candida albicans (colonial morphology on Sabouraud‫׳‬s dextrose agar, Gram
stain, Germ test tube and biochemical reactions).
Cryptococcus neoformans, Malassezia furfur & Dermatophytes.
13. Thirteen week:
ELISA , Coomb´s test, Haemagglutination.
Note:
There will be a presentation subject on Mycology discussed every week by one or two students
presented by power point & on hard copy.
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Course objectives divided according to course work by weeks for
Medical Microbiology (BIOM 322)
First week:
Upon completion of this laboratory, the student will be able to:
1) Descripe and apply with Microbiology Lab Safety Rules.
2) Distinguish different sterilization techniques.
3) Discuss preparation ,classification & utilization of routine primary media.
Second week:
Upon completion of this laboratory, the student will be able to:
1) Explain the use of differential and selective medium.
2) Identify the normal microbial flora.
Third week:
Upon completion of this laboratory, the student will be able to:
1) Explain the medical significance of Staphylococci species.
2) Identify laboratory procedures designed to differentiate among the major Staphylococcal
species.
Fourth week:
Upon completion of this laboratory, the student will be able to:
1) Differentiate among major Staphylococcal species utilizing macroscopic, microscopic,
biochemical reactions and by chemotherapeutic agents.
2) Explain and perform the disc –agar diffusion technique for determination of different
chemotherapeutic agents.
Fifth week:
Upon completion of this laboratory, the student will be able to:
1) Explain the medical significance of Streptococcal species.
2) Identify laboratory procedures designed to differentiate Streptococci on the basis of their
hemolytic activity and biochemical patterns associated with the Lance field group classification.
Sixth week:
Upon completion of this laboratory, the student will be able to:
1) Differentiate among major Streptococcal species on the basis of their hemolytic activity.
2)Select laboratory procedures that differentiates between Streptococcus pneumoniae and other
hemolytic Streptococci .
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Seventh week:
Upon completion of this laboratory, the student will be able to:
1) Explain the medical significance of the family Enterobacteriacae .
2) Define lactose fermenter and non lactose fermenter.
3) Identify laboratory procedures used to differentiate between E.coli &Klebsiella (IMVC).
Eighth week:
Upon completion of this laboratory, the student will be able to:
1) Differentiate between E.coli &Klebsiella according to (IMVC) results.
2) Explain the medical significance of Proteus & Pseudomonas as non lactose fermenter.
3) Identify laboratory procedures used to differentiate between Proteus & Pseudomonas.
Ninth week:
Upon completion of this laboratory, the student will be able to:
1) Differentiate between Proteus & Pseudomonas according to biochemical reactions results.
2) Explain the medical significance of Salmonella &Shigella .
3) Descripe laboratory procedure for TSI test.
Tenth week:
Upon completion of this laboratory, the student will be able to:
Differentiate various TSI different results.
2) Prform laboratory procedures designed to identify family of Enterobacteriacae using
commercial multi test Microsystems.
3) Demonstrate slides of:a) Mycobacterium tuberculosis .
b) Clostridium tetani .
c) Loffler´s serum medium.
d)L.J. medium.
e) Cooked meat broth medium.
f)Anaerobic gas pack system.
Eleventh week:
Upon completion of this laboratory, the student will be able to:
1) Explain medical significance of Campylobacter ,Haemophylus influenzae &Neisseriae .
2) Design scheme of identification of isolated unknown organism.
Twelveth week:
Upon completion of this laboratory, the student will be able to:
1) Explain medical significance of fungi.
2) Identify laboratory procedures used to investigate Candida albicans.
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Thirteenth week:
1) Explain medical significance of Elisa ,Comb's test &haemagglutination test .
2) Identify laboratory procedures to perform these tests and analyze the results .
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Microbiology Lab Safety
Safety DO’s:
Keep
your
Safety DONT’s:
workbench
neat
and Place cultures near the edge of the
organized.
bench.
Label all cultures/containers.
Mix unknown chemicals.
Read
Bergey’s
Manual
to
obtain Think that a nice sounding name for
information about a new organism.
an organism means it harmless.
Ask how to discard used cultures.
Pour any live culture down the sink
drain.
Wear your safety glasses at all times.
Take off your safety glasses or touch
your face with soiled latex gloves .
Report accidents to the instructor Attempt to clean up a spill by yourself
immediately.
or leave the lab to treat an injury by
yourself.
Take a rest break now and then.
Be in a rush to Finish.
AN EXPERIMENT DONE WELL IS…
AN EXPERIMENT DONE SAFELY.
The Microbiology Lab Safety Rules :
Hazards exist in the lab for toxic chemicals and microorganisms. Microbes, in particular have a
great versatility to grow and proliferate in different environments (i.e. opportunistic pathogens),
and thus it is imperative that you conduct yourself safely at all times when in the laboratory. If
the lab instructor becomes concerned for your safety, or the safety of others, you will be asked to
leave the lab.
1. Note the location for the emergency exits, first aid kit, fire extinguisher, Hand wash
station, emergency showers, and eye wash apparatus.
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2. Do not smoke, eat or drink in the laboratory, or place any object on near your mouth.
Keep your books, laboratory manual and workbook at a reasonable distance from your
work area.
3. If a fire starts, or the fire alarm sounds, unplug any electrical apparatus and vacate the
laboratory in an orderly manner.
4. Practice good aseptic techniques by performing the following before starting each class:
(i)
Long hair should be tied back.
(ii)
Wear closed footwear to protect the feet.
(iii)
Wear a clean lab coat.
(iv)
Wear protective glasses at all times.
(v)
Wash your hands thoroughly with soap and water before starting your
exercises.
(vi)
Wear disposable latex gloves when handling blood products (e.g.) whole blood,
plasma, serum, etc.).
5. Do not place any hazardous or infectious materials in the sink. Do not dispose of any solid
material in the sink.
6. To clean up spills of microbial cultures, first contain the spill by placing a paper towel
soaked in 70% ethanol over the spill area. Keep the towel on the spill for 20 minutes. In
form your instructor of the spill. Place the towel in an autoclave (biohazard) waste bag
provided. Ensure you wash your hands immediately after dealing with the spill.
7. Never place any instruments or materials into your mouth. i.e. Do not pipette by mouth.
Use the mechanical apparatus provided.
8. Place pipettes that are used during class immediately into the appropriate waste
container.
9. Place used glass slides and cover slips in glass dishes of disinfectant. Do not discard any
demonstration slides.
10. Place all used bottles, tubes and cultures in the containers provided on each bench for
staff to remove and autoclave.
11. Place contaminated waste in the autoclave bag-lined containers. All other (non-biohazard
waste) can be disposed of in approproiately labeled containers.
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12. All materials requiring incubation or refrigeration must be appropriately labeled and
placed on the trays provided.
13. At the conclusion of each class clean the microscope provided for you. Use lens tissue to
clean the oil immersion lenses and leave the microscope standing with the low power lens
in place. Avoid jolting the microscope.
14. Decontaminate your work bench at the end of each exercise by applying an antiseptic
wash.
15. Before leaving the laboratory for a coffee, lunch a nature break, or at the end of the day,
wash your hands thoroughly with antiseptic soap and water.
Preparing culture media&dispensing
As all other living organisms microorganisms require certain basic nutrients and physical factors
for continuity of life.
Understanding these needs is necessary for successful cultivation of microorganisms in the
laboratory.
Nutritional needs:
These needs are supplied through a variety of media.
1-Carbon:
A-Autotrophy: use inorganic carbon in the form of carbon dioxide.
B-Heterotrophs: can not be cultivitated in a media contain inorganic compounds, they must be
supplied with organic nutrient, primarily glucose.
2- Nitrogen:
Essential atom in protein –nucleic acid synthesis.
3-Non metallic element: a-Sulfur containing amino acid.
b- Phosphorus: formation of nucleic acid DNA and RNA, ATP.
4-Metalic element: Ca++,Zn+,Na+,K+,Cu++,Mn++,Mg++,Fe+2,3 necessary for performance
of varied cellular activities.
5-Vitamins: Contribute to cellular growth and essential in minute conc. For all activities.
6-Water: All cell require water
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7-Energy:
Phototrophs: These use radiant energy.
Chemotrophs: These depend on oxidation of chemical compounds as energy source.
Physical factors:
1-Temperature: Optimum temp. For enzymatic activities 20-40c.
Low temp______________ decrease enzyme activity.
High temp.______________Coagulation of enzymes
2-pH of the extra cellular environment:
Optimum pH is in the neutral range of 7 either increases or decrease pH will slow down the rate
of chemical reactions.
3-The gaseous requirement: O2 in most cells plays a vital role in ATP formation.
Classification of media
1- according to the physical state:
a- Liquid as: peptone water
Nutrient broth
b- Solid: Nutrient agar
C-Semi solid: 0.5% only agar.
2- According to type of media:
a- Simple media: Nutrient broth
Nutrient agar.
b- Enriched media: blood agar
Chocolate agar
c- Selective media: (L.J.) Lowenstein Jensen medium
- Desoxycholate citrate agar (DCA).
- T.C.B.S: for V.cholera.
c- Differential (indicator) medium: Mac.conkey: differentiate between lactose fermented
&non lactose fermented.
e- Sugar media: bacteria vary in sugar fermentation so this media used for differentia.
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Artificial media
Media that will be prepared this session are:
1-Nutrient broth
2-Nutrient agar
3-Mac conkey agar: It is composed of:
*peptone as a nutrient.
*agar as a solidifying agent.
*lactose as test sugar.
*neutral red as indicator that changes pink in the presence of acid which is produced as result
of lactose fermentation.
*bile salts inhibit non intestinal bacteria.
*it differentiates between lactose fermented &non lactose fermented.
Dispensing of the media
All used media should be collected as bio hazards autocalved before get rid
Preparing Culture Media
Necessary skills
Knowledge of the metric system of measurement
Warning: Never pour liquefied agar media down the drain. They solidify in the pipes.
Material :
(This exercise is designed to be performed by students working in pairs.):
At least 1.5 gm of peptone per pair
At least 0.9 gm of beef extrac per pair
At least 300 ml of deionized and / or distilled water per pair
1 Test tube per class containing 15 ml of colored water, marked “15 ml control”
1 Test tube per class containing 10 ml of colored water, marked “10 ml control”
1 500 ml Graduated cylinder per group
1 Balance per group
1 500 ml Erlenmeyer flask per pair
1 Tongue depressor per pair
3 Spatulas per pair
At least 3 weighing papers per pair
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1 Hot plate stirrer and magnetic stirring bar; or 1 ring stand, 1 piece of wire gauze, 1 Bunsen
burner, and 1 stirring rod per pair
1 Magnetic stir bar retriever par class, if necessary 18 Test tubes per pair, all with equal
diameter, at least 16 /150 mm, clean with caps
Flask tongs or other devices for handling hot Erlenmeyer flasks
1 Plastic foam stopper per pair, used on flask (optional)
1 Paper bag per pair, used on flask with stopper (optional)
1 Scissors per group to trim bag of foil
1 Piece of aluminum foil per pair to cover mouth and neck of flask instead of stopper and bag
9 Sterile Petri plats per pair
Baskets or racks to collect and autoclave slants, broths, and pours prepared by students (labeled
“Slants,” ”Broths,” ”Pours”).
1Dispensing apparatus per class; see figure 16.1
Support and base
Funnel
Support ring elamped to stand
Utility clamp
Pinchcock clamp
Rubber tubing
1 Large container of cool water per class
Water baths, 50 oC, (enough surface area to hold 1500 ml Erlenmeyer flask per pair of students.
Keep the water level very low so flasks do not float.)
Enough slant trays to hold 6 slants per pair pH Equipment (per class):
1 L of 1 M NaOH with pipet and pipettor
1 L of 1 M HCl with pipet and pipettor pH Meter of papers
500 m Bottle of pH 7 buffer solution
10 Beakers, 20 ml
Wash bottle of distilled and/or deionized water
Box of Kimwipes or other lint-free tissue
Labeled containers for storage of media in Petri plates
Access to a current DIFCO Manual
Access to the Manual of BBL Products and Laboratory Procedures (optional)
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Primary Objective :
Prepare broths, slants, and plates of media for use in the microbiology laboratory.
Other Objectives:
1. Describe two uses for each form of culture media: solid, semisolid, and liquid.
2. List five functional categories of media; describe the purpose of each and give an example
of each.
3. Distinguish between a chemically defined medium and a chemically complex one.
4. Name two commercial sources of dehydrated culture media.
5. Define: infusion, extract, digests.
6. List the basic nutritional requirements of all bacteria.
7. Summarize attributes of agar that make it and adequate solidifying agent for the
microbiology laboratory.
8. Wash glassware correctly for culture media preparation.
9. Discuss the advantages of utilizing a hot plate stirrer for preparing culture media.
10. Check the pH of a solution.
11. Explain why Petri plates of media are poured at a holding temperature of about 50 oC
instead of 100 oC.
12. Demonstrate knowledge of proper media storage techniques.
Introduction
For this exercise you prepare, dispense, and sterilize culture media (nutrient solutions) for your
own use in later experiments.
Classification of Media
Microbiological media are solid, semisolid, or liquid, each type of medium has its own uses. For
separating bacteria and allowing them to grow into isolated colonies, or for maintaining stock
cultures, a solid surface is utilized. To see if bacteria are motile or not, a semisolid medium can be
employed. Medical specimens containing bacteria are often transported from the site of collection
to the laboratory in semisolid media. Large quantities of microbes are generally cultivated in
liquid media. Also, fermentation and other biochemical tests are often performed in tubes of
liquid media.
Media are categorized by their function. An all purpose medium, such as Tryptic Soy Agar,
supports the growth of most bacteria cultured in the laboratory. Selective media inhibit the
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growth of some microbes. Mac-Conkey’s Agar, for example, inhibits gram-positive bacteria. The
biochemical products of some bacteria reach with differential media, creating a visible result in
the colony or the medium. For example, bacteria that form hydrogen sulfide blacken Triple
Sugar Iron Agar. Enrichment media contain nutrients that favor the growth of certain
microorganisms. Selenite Broth enriches members of the Salmonella group so that they multiply
faster than other fecal organisms and soon outnumber their competitors.
Another function of certain specialized media is conveyance, Transport media are designed to
deliver living microbes from the collection site to the laboratory. Transport Medium Amies, for
example, contains charcoal to adsorb bacterial waste products. This purification helps the
organisms survive in transit.
Media Ingredients
Media are divided into two categories according to their components. If the exact ionic
concentration of every chemical in the medium is known, the solution is called a chemically
defined or synthetic medium. These media are often expensive and time consuming to prepare;
see table 1.1 .
More commonly, ingredients such as beef extract and peptone (partially digested protein) are
included in nutrient recipes; see table 16.2. Media containing these more natural ingredients are
called chemically complex or non-defined. While there are precise recipes for chemically complex
media, no one knows the exact concentration of each ion, Na+ or Cl- for example, because this
amount varies slightly between batches of natural ingredients.
Many dehydrated media – standardized media prepared in large batches, desiccated, bottled,
and sold – are available to microbiologists though companies such as DIFCO Laboratories and
Baltimore Biological laboratory. Both of these companies publish reference manuals that you
investigate to complete this exercise.
Before dehydrated media were widely available and occasionally even today, microbiologists
prepared their own infusions (substances steeped in water). Extracts (concentrated solvents that
have removed active ingredients from animal or vegetable matter). And digests (organic matter
made soluble with heat and moisture).
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Table 1. 1 A Chemicallly Delined Medium, Amino Acid Assay Medium (formula per liter)
Dextrose
50 gm
Guanine hydrochioride
0 02 gm
Sodium acetate
40 gm
Uracil
0.02 gm
Ammonium chloride
6 gm
Xanthine
0. 02 gm
DL-Alanine
0.4 gm
Thiamine hydrochloride
0 .001 gm
L-Arginine hydrochloride
0.484 gm
Pyridoxine hydrochloride
0. 002 gm
Asparagine
0.8 gm
Pyridoxamine hydrochloride
0.0006 gm
L-Aspartic acid
0.2 gm
Pyridoxal hydrochloride
0 0006 gm
L-Cystine
0.1 gm
Calcium pa^tothenale
0.001 gm
L-G!ulamic acid
0. 6 gm
Ribollavin
0 001 gm
Glycine
0.2 gm
Nicotinic acid
0.002 gm
L-Histidine hydrochioride
0.124 gm
p-Aminobenzoic acid
0.0002 gm
DL-Phenylalanine
0 2 gm
Biotin
0 000002 gm
L-Proline
0-2 gm
Folic acid
0.00002 gm
DL-Serine
0.1 gm
Monopotassium phosphate
1. 2 gm
DL-Threonine
0.4 gm
Dipotassium phosphate
1. 2 gm
DL-Tryptophane
0 08 gm
Magnesium sulfate
0 .4gm
L-Tyrosine
0.2 gm
Ferrous sulfate
0 .02gm
DL-Valine
0.5 gm
Manganese sulfate
0 .04 gm
Adenine sulfate
0. 02 gm
Sodium chloride
0 .02 gm
'This medium, available Irom D1FCO Laboialories, is a base for microbiological assay of the ammo
acids leucine, methionine, lysine, or isoleucine. From the DIFCO Manual, 10th ed., 1984. DIFCO
laboratories. Detroit, Ml 48232.
Table 1 .2. A Chemically Complex Medium, Nulrient Agar (formula per 1,000 ml)
Beef extract
3 gm
Peptone
5 gm
Agar
15 gm
From the DIFCO manual 10th ed . 1984. DIFCO Laboratories. Detroit. Mi 48232
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While the nutritional requirements of bacteria vary tremendously between species, all must
have water, carbon, an energy source, nitrogen, and minerals including phosphorus, sulphur,
and trace elements (metal ions). Frequently, growth factors such as vitamins and nucleosides
are also required. And each species has its own optimal pH range.
Notice that agar is not listed with the nutritional requirements of bacteria. This is because it
almost never supplies nutrients. Very few microorganisms can utilize as a carbon source this
complex carbohydrate extracted from red algae. Agar is a solidifying agent used to physically,
not nutritionally, support bacterial growth. Performing in low concentrations of less than 2.0%—leaving room for peptones, carbohydrates, dyes, and so on—agar forms a firm, transparent
gel with most nutrient solutions.
Other attributes of agar also make it an excellent solidifying agent for the microbiology
laboratory. Agar liquifies at about 100° C and remains liquified until its temperature falls to
about 42° C. Thus microbes can be suspended in warm, liquified medium to separate the cells.
After the medium has cooled and solidified, the bacteria can be incubated at temperatures up
to 100" C in this same medium. It remains gelled and the microbes, if adequately diluted, may
grow into individual, isolated colonies.
Preparing Media
When you prepare microbiologies media, cleanliness, accuracy, and sterility are your primary
concerns. All glass-ware should be thoroughly washed. Use deionized or distilled water for
the final rinse. Watch: for cracks that might cause breakage in the autoclave.
The balance is a precision instrument for weighing. Whether you employ a small school
scale or an analytical balance costing thousands of dollars, carefully observe Your instructor's
demonstration of its operation and use the device properly. Remember that you will be
measuring small amounts; 1 gram equals about 1/28 of an ounce.
For media preparation, use only distilled or deionized water. When preparing agar
solutions, utilize a flask that is at least twice the final volume of your recipe. Most ingredients go
into clear solution when swirled in cool water. But some, including agar, require boiling. When
heating the medium, stir it constantly to prevent scorching the bottom.
A hot plate stirrer, which heats your solution while its magnetic stirrer motor spins a tefloncoated magnetic spinbar in your medium, frees you from the time-consuming task of
stirring. But check the solution frequently; agar media tend to boil suddenly, overflowing the
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flask and making a mess of the hot plate and bench top. Have a pair of tongs or other device
available for quickly removing your boiling solution from the heat source.
If you are preparing dehydrated culture media, there is generally no need to check the pH
of the solution. It has already been carefully adjusted. Other liquid media should be compared,
at about room temperature, to a standardized buffer solution, a liquid with a stable pH that
has been adjusted to a set value. Your instructor will demonstrate how to use a pH meter or
pH lest papers.
Media are dispensed into tubes or flasks before sterilizing, but in the autoclave (a sterilizer
employing superheated steam under pressure) media boil-out of Petri dishes. So, plates are
poured after autoclaving, utilizing sterile media and sterile Petri dishes.
Before pouring plates, the tubes or flasks of media are brought down from boiling to a
holding temperature of about 50° C. The tempered medium is easier to handle, and agar at a
suitable pouring temperature does not cause excessive condensation to form inside the lid of
a Petri plate.
When the agar in a Petri dish has cooled sufficiently to solidify, the plates are inverted for
storage. In this position any water from condensation helps hydrate the medium.
Prepared, dehydrated media are stored at the temperatures indicated on the product
description. This information is generally given on the bottle label. The Nutrient Agar you
prepare is best stored between 15° and 30° C. Plates are stored upside down. Placing them
back into their plastic sleeves or in other moisture-proof containers helps prevent desiccation
of the medium. Use the media as soon as possible.
PROCEDURES
EXERCISE :
Preparing Nutrient Broth
a* Working in pairs you prepare a total of 300 ml of Nutrient Broth. You add agar to 200 ml
of the broth, making Nutrient Agar. You dispense the media as follows; see figure l.2:
Broth
10 broths, 10 ml each = 100 ml of broth in 10 lubes
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Agar
2 pours, 15 ml each = 30 ml of agar in 2 tubes (to be poured
into 2 Petri plates after autoclaving
and tempering) 60 ml ol agar in 6 tubes (to be cooled in a
6 slants, 10 ml each = 60 ml of agr in 6 tubes (to be cooled in a slanted position after autoclaving, as
illustrated)
1flask, 110 ml = 110 ml of agar (to be dispensed into 6 or 7 Petri plates after autoclaving and
tempering).
b* Assemble the ingredients you and your partner need to prepare 300 ml of Nutrient Broth:
peptone and beef extract.
c* Assemble the equipment you need to apportion the ingredients: graduated cylinder, 500
ml; balance; tongue depressor; two spatulas; weighing papers, two per pair.
d* Assemble the necessary glassware and accessories: a 500 ml Erlenmeyer flask and a test
tube rack with 18 clean test tubes with closures. All test tubes must be of equal diameter.
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e* If you have access to a hot plate stirrer, you need a magnetic stirring rod. Otherwise, you
can employ a standard hot plate or a ring stand and Bunsen burner to heat your medium.
f* One liter (1,000 ml) of Nutrient Broth contains 3 gm of beef extract and 5 gm of peptone.
How much of each do you need to prepare 300 ml of the medium?
Beef extract
Peptone
Check with your instructor to be sure that your c a l culations are correct.
g* Using the 500 ml graduated cylinder, obtain 300 ml of distilled and/or deionized
water. Dispense about 100 ml into the Erlenmeyer flask.
h* Fold a weighing paper twice. Weigh the weighing paper. Calculate the total of its
weight plus the weight of the required peptone.
i* Use a clean spatula to dispense the correct amount of peptone onto the paper. Add the
powder to the water in your flask. Close the lid of the peptone bottle tightly and return the
bottle to the class supply.
j* Place a twice-folded weighing paper and then a tongue depressor on the balance.
Calculate their total weight plus the weight of the required beef extract.
k* Using a clean spatula for assistance, dispense the correct amount of beef extract onto
the wooden blade. Add the beef extract to the water in your flask, using the blade and stirring
rod to stir it into solution. Close the lid of the beef extract container and return it to the class
supply.
l* If the extract and powder do not go into solution quickly, heat the solution gently,
stirring constantly.
m* Mix in the remaining deionized and/or distilled H2O.
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n* If you have heated your solution, rapidly cool it to about 22° C in a container of
cool water.
o* Then, as demonstrated by your instructor, check the pH of your solution. It should be
6.8 ± 0.2. If necessary, adjust the pH with 1M HC1 and 1M NaOH.
p* Pour 100 ml of Nutrient Broth back into your graduated cylinder.
q* Using the class dispensing apparatus, pour the 100 ml of broth into 10 tubes, 10 ml
each. Be sure all tubes have approximately equal diameters. Match the height of the broth in
your tubes to the 10ml control tube of colored water. Cap the tubes of Nutrient Broth. Loosen
screw caps one-quarter turn. For sterilization, add your tubes to the class baskets marked
"Broths."
Note: For sensitive, biochemical tests requiring precisely measured amounts of reactant, media
is measured into each test tube with a pipet, syringe, or graduated cylinder. This is not
generally required with the growth medium Nutrient Agar.
•
Follow your instructor's directions regarding return or disposal of the cylinder
and other supplies.
Preparing and Dispensing Nutrient Agar
First Laboratory Session
a* Nutrient Agar is prepared with 1.5% agar. You now have 200 ml of Nutrient Broth in an
Erlenmeyer flask.
How much agar should you add? Collect a jar
of agar, a weighing paper, and a clean spatula.
b* Check with your instructor to be sure your calculation is correct, then weigh out the
appropriate amount of agar onto a twice-folded weighing paper.
c* Add the agar to your Nutrient Broth. Return the tightly closed jar of agar to the class
set.
d* Obtain a magnetic stirrer or a s t i r r i n g rod. Use it to agitate the medium constantly
while heating the liquid to boiling over medium heat. Avoid charring the medium with high
heat.
e* Have a pair of flask tongs, a folded paper towel, or other device handy to remove the
Erlenmeyer flask from the heat as soon as the medium boils.
f* When the medium boils, remove it from the heat.
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g* Using the class dispensing apparatus, pour about 30 ml of agar into 2 tubes, 15 ml each.
Be sure all tubes have approximately equal diameters. Match the height of the agar in your
tubes to the 15 ml control tube of colored water. Do not allow Nutrient Agar to cool below 45°
C and solidify in the dispensing apparatus. Cap the tubes of Nutrient Agar; loosen screw caps
one-quarter turn. For sterilization, add your media to the class baskets marked "Pours."
g* Using the dispensing apparatus again, pour about 60 ml of agar into 6 tubes, 10 ml
each. Be sure all tubes have approximately equal diameters. Match the height of the agar in
your tubes to the 10 ml control tube of colored water. Do not allow cooled, solidified Nutrient
Agar to clog the apparatus. Cap the tubes of nutrient Agar; loosen screw caps one-quarter
turn. For sterilization, add the tubes to the class baskets marked "Slants."
i* Follow your instructor's directions for returning the stirring rod. A magnetic stirrer is
autoclavable. It can remain in the bottom of the flask, or you may remove it with a stir bar
retriever.
j* Cover both the mouth and neck of the flask loosely with aluminum foil or utilize a foam
rubber stopper and a trimmed paper bag. Label the flask.
Place the flask with the rest of the class media for sterilization.
k* Your instructor will see that the Nutrient Agar and Nutrient Broth prepared by the
class are autoclaved.
l* The instructor will incubate two tubes of non-sterilized Nutrient Broth, one at room
temperature and the other at 35° C until the next laboratory session. In your laboratory
report, describe what you expect to see in the nonsterilized medium.
Second Laboratory Session
* Compare the appearance of autoclaved and nonsterilized Nutrient Broths. Note your
observations in t h e laboratory report.
Utilizing the DIFCO Manual and/or the Manual of BBL Products and Laboratory Procedures
(Optional)
* While the Nutrient Broth and Nutrient Agar are being sterilized, utilize your laboratory
time to familiarize yourself with the DIFCO Manual and/or the BBL Manual of Products and
Laboratory Procedures. These standards, culture media reference works are found in
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microbiology laboratories worldwide. Use either or both of the manuals to answer the
following questions in your laboratory report.
(i) What percentage of agar is included in each of these media: Nutrient Agar, Mueller
Hinton Agar, Motility Test Medium, And Fluid Thioglycollate Medium? What is the
purpose of each of these media?
(ii) What carbon source(s) is/are present in each of these media: Simmons' Citrate Agar,
Triple Sugar Iron Agar? Which of these media is chemically defined; which is
chemically complex?
(iii) What is the final pH of these media: Sabouraud Dextrose Agar, Brain Heart Infusion?
Which microbes grow best on each of the two media?
(iv) Find and examine the list of pH indicators in your manual(s). Name 5 pH indicators;
describe their color changes and pH ranges. List 5 standard culture media, each of
which contains a different one of your selected pH indicators.
Dispensing Sterile Agar Media
* When fluids are removed from the autoclave, they are superheated and may boil over if
agitated. Do not jar them.
* Place the 2 pours (deeps) and 1 flask in a 50° C water bath.
* Place the 6 slants in a slant tray or other device where they tilt at an angle. This allows
a large surface area to form. Do not allow the medium to spill out the top of the test tubes.
* Take 2 sterile Petri dishes and label the outside of the bottoms (smaller diameter halves)
of the plates. Your name, the date, and the type of medium Nutrient Agar). Use care to avoid
contaminating the sterile plate.
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* When the pours have cooled to about 50cْ remove 1 from the water bath. You now
have about 2 5 minutes before the agar medium solidifies, so work fast .
Dry the bottom of the tube so it does not drip .Briefly flame the neck of the test tube.
Working with your hands near the flame and your nose away from it, quickly and carefully
pour the contents of the tube into the bottom of a sterile, labeled Petri plate. Use the lid of the
plate shield the surface of the dish from your breath. Do not allow agar to splash out of the
dish.
Gently rotate
* Gently tilt and rotate the dish so the medium completely covers the surface. Again, avoid
splashing.
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*If there are any bubbles on the surface of the medium, burst them quickly with the flame, as
in the illustration. Warning: Touch only the base of your Bunsen burner-the stem is hot!
*Repeat steps with another tcmpered tube of Nutrient Agar.
* Follow your instructor's directions concerning recycling of the empty, used test tubes and
closures.
* Label the bottoms of an additional 6 or 7 sterile Petri plates.
* Obtain a flask of Nutrient Agar cooled to pouring temperature. Wipe the bottom of the flask.
* Without touching the mouth of the flask, remove the closure. Briefly flame the lip.
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* Keep the flask tilted from this point on until you have completed pouring all your plates (see
the illustration).
* If you are right-handed, fill your plates from left to right to avoid scooping air into the flask.
While avoiding splashes and working with your hands near the flame and your nose away from
it, quickly pour enough agar into a plate to cover its bottom surface almost completely.
* After pouring and closing the first plate, and before pouring the next one, gently tilt and
rotate the partially filled Petri dish so the agar spreads out over the entire surface.
* Repeat the pouring and tilting process until all plates are poured.
* Holding the base of your Bunsen burner, quickly flame the surface of any plate with bubbles.
* Follow your instructor's directions concerning recycling of the empty, used flask and its
closure.
* When all plates and tubes of Nutrient Agar have solidified, invert the plates and set the slants
in an upright position. Tighten screw caps. Follow your instructor's directions concerning
storage of the culture media. You grow bactcria in these agars and broths in future laboratory
sessions.
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STERILIZATION
Could be divided into: a-physical means
1-heat: a-Dry heat
Red heat
Hot air oven
Incineration
B-Moist heat
Heat below 100ْc
At 100ْc
Above 100 cْ (Autoclave)
2-Radiation
U.V
Ionizing radiation
3-Filtration
Membrane filter
Vacuum filter
Syringe filter
Air filter
B-Chemical means:
1-Phenol
2-Ethyl alcohol
3-Chlorine
4-Hydrogen peroxide
Moist heat
Below 100ْc: as in pasteurization of milk 72c for 20 sec
At 100ْc: boiling for 10 min. as for glass or partly metals as scalpels, forceps.
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Above 100ْc: The autoclave
When water is heated in a closed vessel under pressure the boiling point of water rises above
100ْc.This is the principle of both pressure cooker used for rapid cooking and autoclave.
In autoclave water is heated under double atmospheric pressure and the boiling temp is 120ْc.
Heating articles at this temp. For 20-30 min. is the most efficient method of sterilization. This is
because of:
1-The high temp. It attains.
2-The high penetrating power of steam under pressure.
The simple autoclave
Is a metal cylinder with a closely fitting lid? The lid is connected to a steam discharge tap, a
safety valve and a manometer.
Direction for use:
1-Water is placed in the bottom and the articles to be sterilized are placed on top of a perforated
tray above the water level and the lid is tightly closed.
2-The safety valve is adjusted to 15 ib. /in2 = double atmospheric pressure.
3-When the steam pressure reaches the desired level (2 atmospheric) the safety valve will allow
excess steam to escape.
4-From this point the sterilization timing which is 20-30 min is calculated.
5-Then the heater is turned off, and the autoclave is allowed to cool down before opening the lid.
Uses:
1-Sterilization of surgical instruments, bed linen, surgical dressing gowns, cotton, gauze, and for
any culture media not destroyed by heat.
For best results:
1-Don not over load the autoclave for better penetration of steam and contact with articles.
2-Start timing from the moment the manometer read 2 atmospheric pressure.
Use of Differential and Selective Media
3- Don not opens the lid before pressure goes down to atmospheric pressure.
To test efficiency:
1- Efficacy of dried bacterial spore killed at 120ْc.
2- Chemical indicator.
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Use of Differential and Selective Medium
PURPOSE
To become familiar with the use and function of specialized media for selection and differentiation of microrgamsms.
PRINCIPLE
Numerous special-purpose media are available for functions such as:
1. Isolation of bacterial types from a mixed population of organisms.
2-Differentiation among closely relatcd groups of bacteria on the basis of macroscopic
appearance of the colonies and biochemical reactions within the medium.
3. Enumeration of bacteria in sanitary microbioiogy. Such as in water and sewage. And also in
food and dairy products.
4. Assay of naturally occurring substanccs such as antibiotics. vitamins. and products of industrial fermentation.
5. Characterization and identification of bacteria by their abilities to produce chemical changes
in different media.
In addition to nutrients necessary for the growth of all bacteria, special-purpose media
contain one or more chemical compounds that are essential for their functional specificity. In
this exercise, two types of media will be studied and evaluated.
Selective Media:
These media are used to select (isolate) specific groups of bacteria. They incorporate chemical
substances that inhibit the growth of one type of bacteria while permitting growth of another,
thus facilitating bacterial isolation.
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Differential Media :
These
can
distinguish
among
morphologically
and
biochemically related groups of organisms. They incorporace chemical compounds that, following inoculation and incubation. produce a characteristic change in the
appearance of bacterial growth and/or the medium surrounding the colonies, which permits
differentiation.The following media. which are representative of these two types, will be
investigated in this exercise:
1. Mannitol salt agar: This medium contains a high salt concentration. 7.5%NaCl. which is
inhibitory to the growth of most bacteria other than the Staphylococci. The medium also
performs a differential function: It contains the carbohydrate mannitol. Which some
Staphylococci are capable of fermenting, and phenol red a pH indicator for detecting acid
produced by mannitol-ferminting Staphylococci. These Staphylococci exhibit a yeilow zone
surrounding their growth; Staphylococci that do not ferment mannitol will not producc a change
in coloration.
2-Blood agar: The blood that is incorporated into this medium is an enrichment ingredient for
the cullivation of fastidious organisms such as the Strepcococcus sp. The blood also permits
demonstralion, of the hemolytic properties of some microorganisms, particulary the Streptococci,
Whose hemolytic activities are classified as:
a. Gamma hemolysis: No lysis of red blood cells results in no significant change in the
appearance of the medium surrounding the colonies.
b. Alpha hemolysis: Incomplete lysis of red blood cells, with reduction of hemoglobin to
methemoglobin, results in a greenish halo around the bacterial growth.
c. Beta hemolysis:
Lysis of red blood cells with complete destruction and use of hemoglobin by the
organism results in a clear zone surrounding the colonies. This hemolysis is produced by two
types of bela hemolysins, namely Streptolysin O. an antigenic. Oxygen-labile enzyme, and
streptolysin S, a nonantigenic, oxygen –stable lysin. The hemolytic reaction is enhanced when
blood agar plates are streaked and simultaneously stabbed to show subsurface hemolysis by
streptolysin O in an enviroment with reduced oxygen tension.
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3. MacConkey agar:
The inhibitory action of crystal violet on the growth of gram-positive organisms allows for. The
isolation of gramnegative bacteria. Incorporation of the carbohydrate lactose, bile salts, and
the pH indicator neutral red permits differentiation of enteric bacteria on the basis of their
ability to ferment lactose. On this basis, enteric bacteria are separated into two groups:
a. Coliform bacilli produce acid as a result
of lactose fermentation. The bacteria exhibit a red coloration on their surface. E.coli
produces greater quantities of acid from lactose than other coliform species. When this
occurs, the medium surrounding the growth also becomes red because of the action of the
acid that precipitates the bile salts, followed by absorption of the neutral red.
b. Dysentery, typhoid, and paratyphoid bacilli are not lactose fermenters and therefore do not
produce acid. The colonies appear uncolored and frequently transparent.
Materials
Cultures:
24- to 48-hour trypticase soy broth cultures of Enterobacter aerogenes, Escherichia coli, Streptococcus var. Lancefield Group E. Streptococcus mitis. Enterococcus faecalis, Staphylococcus
aureus, Staphylococcus epidermidis ,and Salmonella typhimurium.
Media:
Per designated student group: one each of mannitol salts agar plate, blood agar plate, MacConkey agar plate.
Equipment
Bunsen burner. inoculating loop, and glassware marking pencil .
Procedure:
Using the bacterial organisms listed in step 2. Prepare and inoculate each of the plates in the
following manner:
a. Appropriately label the cover of each plate ,
as indicated in the section entitled "laboratory Protocol."
b. Divide each of the Petri dishes into the
required number of sections (one section for each different organism) by marking the
bottom of the dish. Label each section with the name of the organism to be inoculated as
illustrated in Figure 2.1
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c. Using sterile technique, inoculate all plates, except the blood agar plate. with the designated
organisms by making a single line of inoculation of each organism in its appropriate
section. Be sure to close the Petri dish and flame the inoculating needle between
inoculations of the different organisms. Refer to Figure 2.1 for an illustration of this
procedure.
d. Using sterile technique. inoculate the blood agar plate as described in step J c. Upon
completion of each single line of inoculation. use the inoculating loop and make three or
four stabs at a 45° angle across the streak.
2. Inoculate each of the different media with the following:
a. Mannitol salts agar: S. aureus, S. epidermidis. Group E, and E. coli.
b. Blood agar: E. faecalis. S. mitis.and Streptococcus viridans. Lancefield Group E.
c. MacConkey agar: E. coli, E. aerogenes. S. typhimurium. and S. aureus.
3.. Incubate the plates in an inverted position for 24 to 48 hours at 37°C.
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Microbial flora of teeth, throat, skin, nail&hair
Microbial flora of the throat and skin include: normal flora
-pathogenic flora
*Normal flora: are heterogeneous population of micro-organisms that inhabit the skin and mucous
membrane of healthy person. They consist of:
1-The resident flora which are relatively fixed types of bacteria and regularly found in a given area.
2-The transient flora are derived from the environment they may remain for hours, days, or weeks but
don't become permanently established.
Function of normal flora:
1-They perform importana metabolic functions e.g:
a-Synthesis of vitamin K.
b-Conversion of bile pigment and bile acids.
c-Absorbtion of nutrients from the intestine.
2-They inhibit colonization and infection by pathogenic bacteria(bacterial antagonism).
Factors which can disturb the normal flora:
1-Anti-microbial agents which lead to super-infection.
2-obstruction
3-Hormonal changes.
**Under certain conditions flora or commensal bacteria may cause disease and are considered potential
pathogen.
Normal flora of skin e.g: Staphylococci(S.epidermidis),Streptococci(α-haemolytic,non
haemolytic).
Normal flora of upper respiratory tract e.g: Staphylococci,Streptococci .
Pathogenic microbial flora:
It is the micro-organism which infect the skin and m.m and cause diseases e.g Staphaureus &Beta
haemolytic streptococci .
Why we use blood agar for growing of microbial flora of throat:
It is enriched media has high nutritive value ,it is an indicator media it can help in identifying bacteria
by their haemolytic action on red cell either the alpha –hemolytic &beta –haemolytic reactions of some
Streptococci& Staphylococci .
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Respiratory pecimens
Throat Specimens
A. Selection. Success with culture or with direct antigen detection depends on
firmly and completely sampling an area of the inflamed throat.
1. Using a tongue blade to hold the tongue down, look at the back of the
throat and the tonsillar area for localized areas of inflammation and exudate.
2. These areas are the most productive for producing cultures of the etiologic
agents of acute pharyngitis.
B. Collection
l. Materials
a. Dacron or calcium alginate swab and transport medium
b. Tongue blade
2. Method
a. Carefully but firmly rub the swab over several areas, of exudate or over
the tonsils and posterior pharynx (Fig. 19).
Figure 19. Firmly sampling only the inflamed areas of the throat and tonsils and avoiding other
oral sites will enhance detection of etiologic agents.
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1. Working with isolated, pure colonies from your TSA plates, Inoculate the
Significant media that you selected and prepared in Activity 2.
2. Label your subcultures carefully, and incubate them at 37ْC for 48 hours or the
appropriate time for the specific tests that you are doing.
After incubation, gather your data, and record them in a self-designed table on the
worksheet for this module. Draw a conclusion from these data as to the genus and species of
your two significant throat organisms, if present. Submit these names in the appropriate place
on your worksheet. Once again, it would be to your advantage to complete this module to the
best of your ability in preparation for unknowns.
Related Experience
If you isolate normal flora organisms only, it is strongly recommended that you identify
them.
Phonetic Pronunciation
Neisseria meningitidis = ny-seer'-ee-uh muh-nin-juh-ty'-dis Haemophilus
influenzae = hee-mof-uh-Ius in-fioo-en'-zee Bordetella pertussis = borduh-tel!' -uh per-tuss' – is
Specimen Collection and Processing
b. Do not touch the cheeks, teeth, or gums with the swab as you withdraw
it from the mouth.
c. Insert the swab back into its packet, and crush the transport medium
vial in the transport container.
C. Labeling
1. Label the swab container with patient identification data, including the
time of collection.
2. Note any antimicrobial agents currently being taken by the patient.
3. Note whether the specimen is for culture or direct antigen detection. Indicate whether it is for throat culture or a streptococcus screen.
4. Indicate the suspected pathogen if other than streptococci, e.g., N.gonorrhoeae.
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D. Transport
1. Transport the swab to the laboratory as soon as possible.
2. If transport is to be delayed beyond I h, refrigerate the swab.
E. Comments
1. A streptococcus screen (culture) will test for and report only the presence or absence of
beta-hemolytic Streptococci, including group A. A throat culture will, in addition to
revealing group A Streptococci, reveal other Beta-hemolytic streptococci, Haemophilus
spp., and significant numbers of other potential respiratory pathogens, including
Streptococcus pneumoniae. Pseudomonas aeruginosa, Staphylococcus aureus, or other
suspected pathogens as agreed upon by the users.
2. Beta-hemolytic streptococci are routinely identified and reported. They are the primary
cause of acute bacterial pharyngitis and need not be tested, for succeptibility to
antimicrobial agents. .
3. Haemophilus spp. may be reported in pediatric patients when requested,
although they are part oi the normal flora in children and adults.
4. Throat culture for N. gonorrhoeae is available on request, but the laboratory must be alerted to the request.
5. Routine susceptibility testing is not routinely done on any isolate from the
Throat, although a screen for methicillin-resistant S. aureus (MRSA) may
be requested if the carrier state for MRSA is suspected.
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Name ------------------------------------Lab Section -----------------------------Experiment 3 throat culture:
Activity 2
Gram stains reaction and cell arrangement of the predominating organism(s):
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Activity 3
Significant media used
Results
Blood agar -------------------------------------------------------------------------------Chocolate agar --------------------------------------------------------------------------Mac Conkey and / or EMB -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Other Tests performed
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Genus of predominating organism isolated: ----------------------------------------
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Identification of Human Staphylococcal Pathogens
PURPOSE
To become familiar with
1. The medical significance of the Staphylococci
2. .Selected laboratory, procedures designed to differentiate among the major staphylococcal
species.
PRINCIPLE
The genus Staphylococcus is composed of both pathogenic and nonpathogenic organisms. They
are gram-positive cocci and occur most commonly as irregular clusters or spherical cells. They
are mesophilic non-spore-formers; however, they are generally highly resistant to drying, especially when sequestered in organic maller such as blood Pus, and tissue fluids. They are capable
of surviving outside of the body for extendcd periods of time, even up to several months. Many
Staphylococci are indigenous to skin surfaces and mucous membranes of the upper respiratory
tract. Breaks in the skin and mucous linings may serve as portals of entry into underlying tissues,
with the possibility of infection by virulent strains.
The three major species include S. aureus,
S. saprophyticus , and S. epidermidis. Strains of the last two species are generally avirulent: however, under special circumstances in which a suitable portal of entry is provided, S. epidermidis
may be the etiological agent for skin lesions and endocarditis, and S. saprophyticuS has been implicated in some urinary tract infections.
Infections are primarily associated with S. aureus pathogenic strains that are often responsible for the formation of abscesses, locailzed pus-producing lesions. These lesions most commonly occur in the skin and its associated structures, resulting in boils, carbuncles, acne, and
impetigo. Infections of internal organs and tissues are not uncommon, however, and induce
pneumonia, osteomyelitis (abscesses in bone and bone marrow), endocarditis (inflammation
of the endocardium), cystitis (inflammation or the ulinary bladder), pyelonephritis inflammation
of the kidneys). Staphylococcal enteritis due to enterotoxin contamination or foods,
and, on
occasion, septicemia.
Strains of S. aureus produce a variety of metabolic end products, some of which may play
roles in the organisms' pathogenicity.
Included among these are coagulase, which causes clot formation: leukocidin, which lyses white
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blood cells: hemolysins, which are active against red blood cells, and enterotoxin, which is
responsible for a type of gastroenterilis. Additional metabolites of a nontoxic nature are DNase,
lipase, gelatinase, and the fibrinolysin staphylokinase.
When there is a possibility or staphylococcal infection. isolation of S. aureus is of clinical
importance. These virulent strains can be differentiated from other staphylococci and identined
by a variety of laboratory tests, some of which are illustrated in Table 3.1
Table 3.1 Laboratory tests for differentoation of staphylococcal species
Test
S.aureus
S.epidermidis
S.saprophyticus
Growth
+
+
+
Fermentation
+
-
-
Colonial pigmentation
Generally golden
White
White
Mannitol salt agar
yellow
Coagulase
+
-
-
Dnase
+
-
-
Hemolysis
Generally beta
-
-
Novobiocin sensitivity
Sensitive
Sensitive
Resistant
In this exercise you will distinguish among the staphylococcal species by traditional test
procedures both. The traditional and computer-based procedures are described below.
The traditional procedures involve the following steps:
1. Mannitol salt agar: This medium is selective for salt-tolerant organisms such as staphylococci. Differentiation among the staphylococci is predicated on their ability to ferment
mannitol Following incubation. Mannitol fermenting organisms, typically S. aurerus strains,
exhibit a yellow halo surrounding their - growth, and non fermenting strains do not.
2. Coagulase test: Production of coagulase is indicative of an S. aureus strain. The enzyme acts
within host tissues to conven fibrinogen to fibrin. It is theorized that the fibrin meshwork that
is formed surrounds the bacterial cells or infected tissues, protecting the organism from
nonspecific host resistance mechanisms such as phagocytosis and the antistaphylococcal
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activity of normal serum. In the coagulase tube test for bound and free coagulase, a
suspension of the test organism in citrated plasma is prepared and the inoculated plasma is
then periodically examined for fibrin formation, or coagulation. Clot formation within 4
hours is interpreted as a positive result and indicative of a virulent S. aureus strain. The
absence of coagulation after 24 hours of incubation is a negative result, indicative of an
avirulent strain.
3. Deoxyribonuclease (DNase) test: Generally. coagulase-positive staphylococci also produce the hydrolytic enzyme DNase thus this test can be used to reconfirm the identification of
S. aureus. The test organism is grown on an agar medium containing DNA Following
incubation. DNase activiry is detemined
by the addition of 1N HCL to the surface of the agar. DNase-positive cultures capable of
DNA hydrolysis will show transluecense around the area of growth. The absence of this halo
is indicative of a negative result and the inability of the organism to produce DNase.
4. Novobiocin sensitivity: This test is used
to distinguish S. epidirmidis from
S. sapro-
phticus
It requires inoculation or a Mueller Hinton agar plate
with the test organism
And application of a 30-ug novobiocin antibioric disc to the surrace of the agar. Following
incubation. The sensitivty of an organism to the antibiotic is determined
by the Kirby Bauer method .
A computer-assisted procedure is the STAPH IDENT system (developed by Analytab
Products division or Sherwood Medical, Plainview, NewYork). STAPH-IDENl is a rapid,
computer-based micromethod for the separation and identification of the newly proposed 13
species of staphylococci. The system consists of ten microcupules containing dehydrated
substrates for the performance of conventional and chromagenic tests. The addition of a
suspension of the test organism serves to hydrate the media and to initiate the biochemical
reactions. The identification of the staphylococcal species is made th the aid of the differential
charts or the API STAPH-IDEINT Profile Register that is part of the system 'Table 3.2), or both.
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Materials
Cultures:
24 hour trypticase Soy agar Slant cluture of Staphylococcus epidermidis. Staphylococcus saprophyticus (ATCC 15305) and Staphylococcus aureus (ATCC e 27660) 24-hour blood agar cultures
of the above arganisms for the STAPH-IDENT system.
Media:
Per designated student group: three mannitol salt agar plates, one deoxyriboncleic acid agar
plate, three Mueller – Hinton agar plates, and the STAPH-IDENT svstem
Reagents:
Citrared human or rabbit plasma, 1n HCL, and McFarland barium sulfate standards.
Equipment:
Bunsen burner. Inoclating loop 13× 100 mm test tubes, sterile Pasteur pipetles, 1-m1 sterile
pipettes. mechanical pipettes, mechanical pipetting device sterile cotton swabs. 30-ug novobiocin
antibiotic discs glassware marking pencil. Forceps and beaker with 95% alcohol.
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Table 3.2 API S"7"APH-IDENT Profile Register
Profile Identification
P rofil e Identification
0040
STAPH CAPITIS
3560
STAPH HYICUS (An)
0060
STAPH HAEMOLYTICUS
3601
STAPH SIMULANS
0100
STAPH CAPITIS
o 140
STAPH CAPITIS
0200
STAPH COHNII
4060
0240
STAPH CAPITIS
4210
STAPH SClURI
0300
STAPH CAPITIS
4310
STAPH SClURI
0340
STAPH CAPITIS
4420
STAPH HAEMOLYTICUS
0440
STAPH HAEMOLYTICUS
4440
STAPH HAEMOLYTICUS
0460
STAPH HAEMOLYTICUS
4460
STAPH HAEMOLYTICUS
0600
STAPH COHNII
4610
STAPH SClURI
0620
STAPH HAEMOLYTICUS
4620
STAPH HAEMOLYTICUS
0640
STAPH HAEMOLYTICUS
0660
STAPH HAEMOLYTICUS
1 000
STAPH EPIDERMIDIS
1 040
STAPH EPIDERMIDIS
1 300
NOVO S
STAPH SAPROPHYTICUS
NOV
STAPH HAEMOLYTICUS
STAPH HAEMOLYTICUS
4700
STAPH AUREUS
COAG+
STAPH SClURI
COAG-
4710
STAPH SClURI
STAPH AUREUS
5040
STAPH EPIDERMIDIS
1 540
STAPH HYICUS (An)
5200
STAPH SClURI
1 560
STAPH HYICUS (An)
5 210
STAPH SClURI
COAG+
5300
STAPH AUREUS
COAG-
2000
STAPH SAPROPHYTICUS
STAPH SClURI
NOVO R
NOVO S
STAPH HOMINIS
5310
STAPH SClURI
2001
STAPH SAPROPHYTICUS
5600
STA?H SClURI
2040
STAPH SAPROPHYTICUS
5610
STAPH SClURI
NOVO R
COAG+
STAPH HOMINIS
5700
STAPH AUREUS
NOVO S
COAG-
2041
STAPH SIMULANS
STA?H SClURI
2061
STAPH SIMULANS
5710
STAPH SClURI
2 141
STAPH SIMULANS
5740
STAPH AUREUS
2 161
STAPH SIMULANS
2201
STAPH SAPROPHYTICUS
6001
STAPH XYLOSUS
ARA+ XYL+
2241
STAPH SIMULANS
STAPH SAPROPHYTICUS
ARA- XYL-
2261
STAPH SIMULANS
6 011
STAPH XYLOSUS
2341
STAPH SIMULANS
6021
STAPH XYLOSUS
2361
STAPH SIMULANS
6 101
STAPH XYLOSUS
STAPH HOMINIS
6 121
STAPH SAPROPHYTICUS
6221
2 400
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2401
STAPH SAPROPHYTICUS
6300
STAPH AUREUS
2421
STAPH SIMULANS
6301
STAPH XYLOSUS
2441
STAPH SIMULANS
6311
STAPH XYLOSUS
2461
STAPH SIMULANS
6321
STAPH XYLOSUS
2541
STAPH SIMULANS
6340
STAPH AUREUS
COAG+
2 561
ST.:l.PH SIMULANS
STAPH WARNER
COAG-
2601
STAPH SAPROPHYTICUS
6400
2611
STAPH SAPROPHYTICUS
6401
2661
STAPH SIMULANS
2721
STAPH COHNII (SSP1)
6421
STAPH XYLOSUS
2741
STAPH SIMULANS
6460
STAPH WARNERI
2761
STAPH SIMULANS
6501
6521
STAPH WARNERI
STAPH XYLOSUS
ARA+ XYL+
STAPH SAp.ROPHYTICUS
ARA- XYL-
STAPH XYLOSUS
3000
STAPH E?IDERMIDIS
6600
STAPH WARNERI
3040
STAPH EPIDERMIDIS
6601
STAPH SAPROPHYTICUS
ARA- XYL-
STA.PIi XYLOSUS
ARA+ XYL+
3 140
STAPH EPIDERMIDIS
3540
STAPH HYICUS (An)
6611
STAPH XYLOSUS
3541
5TAPH INTERMEDIUS(An)
6621
STAPH XYLOSUS
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Tabe, 3.2 (continued)
PROFILE
Identification
Profile
Identification
6700
STAPH AUREUS
7421
ST.APH XYLOSUS
6701
STAPH XYLOSUS
7 501
STAPH INTERMEDIUS (An)
6721
STAPH XYLOSUS
6731
STAPH XYLOSUS
STA:PH
7000
EPIDERMIDIS
STAPH ,XYLOSUS
7521
S"'APH XYLOSUS
7541
SAPH INTERMEDIUS (An)
7560
STAPH HYICUS (An)
7021
STAPH XYLOSUS
7601
STAPH XYLOSUS
7040
STAPH EPIDERMIDIS 7621
STAPH XYLOSUS
7 141
STAPH INTERMEDIUS(An)
7 63'1
STAPH XYLOSUS
7300
STAPH AUREUS
7700
STAPH AUREUS
7321
STAPH XYLOSUS
7701
STAPH XYLOSUS
7340
STAPH AUREUS
7721
STAPH XYLOSUS
7401
STAPH XYLOSUS
7740
STAPH AUREUS
PROCEDURE
I. Preparation of DNA agar plate culture:
a. With a glassware marking pcncil divide the bottom of the plate into three sections. Label
each section with the name of the organism to be inoculated.
b. Aseptically make a single line of inoculation of each test organism in its respective sector
on the agar plate.
2. Preparation of agar plate cultures for novobiocin sensitivity dctermination:
a. Label the three Mueller-Hinton agar plates with the name of the test organism to be
inoculated. Inoculate each plate with its respectivc organism according to the Kirby-Bauer
procedure.
b. Using alcohol-dipped and named forceps. Aseptically apply a novobiocin antibiotic disc to
the surface of each inoculated plate. Gently press the discs down with sterile forceps to
ensure that they adhere to the agar surface.
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3. Preparation of mannitol salt agar plate cultures: Aseptically make a single line of inoculation
of each test organism in the center of the appropriately labeled agar plates.
4. Incubate all plate cultures in an invrted position for 24 to 48 hours at 37ْC
5. Coagulase test procedure:
a. Label three 13 x 100 mm test tubes with the name of the organism to be inoculated.
b. Aseptically add 0.5 ml of a1:4 dilution of citratcd rabbit or human plasma a 0.1 ml of each
test culture to its appropriately labeled test tube.
c. At the end of the laboratory session incubate all tubes that are coagulase-negative for 20
hours at 37°C.
6. STAPH-1DETNT system procedure:
a. Preparation of strip:
i. Dispense: 5 ml of tap water into incubation tray
ii. Place API strip in incubation tray.
b. Preparation of inoculum:
i. Add 2 ml of 0.85% saline (pH 5.5-7.0) 10 a sterile 15 x I50-mm test tube.
ii. Using a sterile swab pick up a sufficient amount of inoculum to prepare a saline
suspension with a final turbidity that is equivalent to a No. 3 McFarland (BaS04)
turbidity standard. Caution: Be sure to use suspension within
15 minutes of preparation.
c. with a sterile Pasteur pipette add 2 or 3 drops of the inoculum to each micro- cupule.
d. Place plastic lid on tray and incubate for 5 hours at 37°C.
OBSERVATIONS AND RESULTS
1. Examine the bacterial plasma suspensions for clot .formation 5 minutes, 20 minutes. 1 hour,
and hours after inoculation by holding the test tubes in a standered position. Place all
coagulase negative suspensions in an incubator at 37ْC for observation 24 hours after
inoculation. Record vour observations and results in the chart.
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Staphylococcal
Appearance of plasma clotted (+) or Unclotted (-)
Coagulase
Species
5 min
(+) or (-)
20 min
1 hr
4 hr.
24 hr.
s. aureus
s. epidermidis
s. saprophyticus
2. Examine the mannitol salt agar plate. Note and record the following in the chart:
a. Presence (+) or absence (-) of growth of each test organism.
b. Color of the medium surrounding the growth of each test organism.
c. Whether each test organism is a mannitol fermentcr (+) or nonferrnenter (-).
3. Flood the DNA agar plate with 1N HCL . Observe for the delayed development of
transluesent clear area surrounding the growth of each test organism. Record your color
observation and indicate the presence (+) or absence (-) of DNase activity in the chart.
4. with a metric ruler measure the size of the zone of inhibition. If present surrounding each of
the novobiocin discs on the agar plates. A zone of inhibition of 17 mm or less is indicative of
novobiocin resistance. Whereas a zone greater than 17mm indicates that the organism is
sensitive to this antibiotic. Record in the chart the susceptibility of each test organism to
novobiocin as sensitive (S) or resistant (R).
Refer to photo for illustration of the reactions :
Procedure
S. aureus
S. epidermidis
S.sapraphyticus
--------------------------------
--------------------------------
----------------------------
--------------------------------
--------------------------------
----------------------------
--------------
--------------
-------------------
--------------------------------
--------------------------------
----------------------------
--------------------
--------------------
----------------------
--------------------------------
--------------------------------
----------------------------
--------------------
--------------------
----------------------
Mannitol salt agar
Growth
Color of medium
Fermentation
DNA agar
Calor of medium
DNase activity
Novobiocin sensitivity
Growth inhibition in mm
Susceptibility-(R) or (S)
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5. Interpret your STAPH-IDENT system reacrions on the basis of the observed color
.changes in each of the microcupules described in the chart below. Record your color
observation and result as (+)
or (-) for each test in this chart.
Microcupule
No
1
PHS
Interpretation of Reactions
Reaction Results
Substrate
Positive
Negative
Color
P-nitrophenyi-
Yellow
Clear
phosphate
or
straw
(+) or (-)
–
colored
Disodium salt
2
3
URE
GLS
Urea
p-nitrophenyl
–
B-D-
Purple to red –
Yellow
orange
orange
Yellow
Clear or
glucopyranoside
MNE
Mannose
Yellow yellow
5
MAN
Mannitol
orange
6
TRE
Trehalose
7
SAL
Salicin
8
GLC
p-nitrophenyl
B-D-
Yellow
glucuronide
9
10
ARG
NGP
Arginine
yellow
Straw – colored
4
–
or
Red or orange
Clear
or
straw
–
colored
Purple to red –
Yellow or yellow –
orange
orange
2- naphthyl – B-D
Add 1-2 drops of STAPH-IDENT
galactopyranoside
reagent
Plum-purple
Yellow or colorless
(mauve)
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6. Construct a four-digit profile for your unknown organism as follows: A four-digit profile is
derived from the results obtaind with STAPH-IDENT. The 10 biochemical tests are divided into
four groups as follows:
PHS
MNE
SAL
URE
MAN
GLC
GLS
TRE
ARG
NGP
Only positive reacactions are assigned a numcrical value. The valuc depends on the location
within the group.
A value of I for the first biochemical in each group (i.e. PHS, MNE . . .)
A value of 2 for the second biochemical in each group (i.e. URE. MAN . . .) Avalue of 4 for
the third biochemical in each group (i.e. GLS. TRE . . .) Avalue Of 0 for all negative
reactions.
Chemical Agents of Control: Chemotherapeutic Agents
Chemotherapeutic agents are chemical substances used in the treatment of infectious diseases,
Their mode of action is to interfere with microbial metabolism, thereby producing a static or
cidal effect on the microorganisms, without producing a like effect in host cells. These drugs can
be separated into two categories:
1. Antibiotics are synthesized and secreted by some true bacteria. Actinomycetes, and fungi that
destroy or inhibit the growth of other microorganisms . Today, some antibiotics are
laboratory synthesized or modified; however, their origins are living cells.
2. Synthetic drugs are synthesized in the laboratory
To determine a therapeutic drug of choice. One must know its mode of "action possible adverse
side effects in the host, and the scope of its antimicrobial activity. The specific mechanism of
action varies among differem drugs, and the short-term or long-term use of many drugs can
produce systemic side effects in the host. These vary in severity from mild and temporary upsets
to permanent tissue damage (Table 11.1)
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SYNTHETIC AGENTS:
Antibiotic
Penicillin
Mode of Action
Possible Side Effects
Prevents transpeptidation of the N-acetyl
muramic acids, producing a weakened
Penicillin
resistance;
sensitivity
(allergic reaction)
peptidoglycan structure.
Streptomycin
Has an affinity for bacterial ribosomes,
causing misreading of codons on mRNA,
May produce damage to auditory
nerve,causing deafness
thereby interfering with protein synthesis
Chloramphenicol
Has an affinity for bacterial ribosomes,
preventing
between
peptide
amino
bond
acids
formation
during
protein
May cause aplastic anemia. which is
fatal because of destruction of RBCforming and WBC-forming tissues
synthesis
Tetracyclines
Have an affinity for bacterial ribosomes;
prevent hydrogen bonding between the
Permanent discoloration of teeth in
young children
anticodon on the tRNA-amino acid
complex and the codon on mRNA during
protein synthesis
Bacitracin
Inhibits cell-wall synthesis
Nephrotoxic if taken internally; used
ror topical application only
Polymyxin
Destruction of cell membrane
Toxic if taken internally; used for
topical application only
Sulfadiazine (sulfonamide) produces a static effect on a wide range of microorganisms by
mechanism of action called competitive inhibi-tion. The active component or the drug. sulfanilamide, acts as an antimetabolite that competes with the essential metabolite, p-aminobenzoic acid
(PABA), during the synthesis of folic acid in the microbial cell. Folic acid is an essential cellular
coenzyme involved in the synthesis of amino acids and purines. Many microorganisms possess
enzymatic pathways for folic acid synthesis and can be adversely affected by sulfonamides. Human cells lack these enzymes, and the essential folic acid enters the cells in a preformed state .
Table 11.1 Prototypic Antibiotics
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Therefore these drugs have no competitive effect on human cells. The similarity between the
chemical structure of the antimetabolite sulfanilamide and the structure of the essential metabolite PABA is illustrated in Figure 11.1
PART A: THE KIRBY-BAUER ANTIMICROBIAL SUSCEPTIBILITY TEST
PROCEDURE:
PURPOSE
To become acquainted with the Kirby-Bauer procedure for the evaluation of the antimicrobial
activity of chemotherapeutic agents.
PRINCIPLE
The available chemotherapeutic agents vary in their scope of antimicrobial activity. Some have a
limited spectrum of activity, being effective against only one group of microorganisms. Others
exhibit broad-spectrum activity against a range of microorganisms. The drug susceptibilities of
many pathogenic microorganisms are known, but it is sometimes necessary to test several agents
to determine the drug of choice.
A standardized filter-paper disc-agar diffusion procedure, known as the Kirby-Bauer
method, is frequently used to determine the drug susceptibility of microorganisms isolated from
infectious processes (Figure 11.21) This method allows for the rapid determination of the
efficacy of a drug by measuring the diameter of the zone of inhibition that results from diffusion
or the agen into the medium surrounding the disc. In this procedure, filter-paper discs of
uniform size are impregnated with specified concentrations of different antibiotics and then
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placed on the surface of an agar plate that has been seeded with the organism to be tested. The
medium of choice is Mueller-Hinton agar, with a pH of 7.2 to 7.4, which is poured into plates to
a uniform depth of 5 mm and refrigerated on solidification. Prior to use, the plates are
transferred to an incubator at 37ْC for 10 to 20 minutes to dry off the moisture that develops on
the agar surface. The plates are then heavily inoculated with a standardized inoculum by means
of a cotton swab to ensure the confluent growth of the organism. The discs are aseptically
applied to the surface of the agar plate at well-spaced intervals. Once applied, each disc is gently
touched with a sterile applicator stick to ensure its firm contact with the agar surface.
Following incubation, the plates are examined for the presence of growth inhibition, which
is indicated by a clear zone surrounding each disc. The susceptibility of an organism to a drug
is determined by the size of this zone, which itself is dependent on variables such as:
1. The ability and rate of diffusion of the antibiotic into the medium and its interaction with
the test organism.
2. The number of organisms inoculated.
3. The growth rate of the organism.
4. The degree of susceptibility of the organism to the anribiotic.
A measurement of the diameter of the zone of inhibition in millimeters is made and its size is
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Compared to that contained in a standardized chart, which is shown in Table 11.2 Based on this
comparison, the test organism is determined to be resistant. intermediate. or susceptible to the
antibiotic.
MATERIALS
Cultures
0.85% saline suspensions of Escherichia coli. Staphylococcus aureus, Pseudomonas aeruginosa,
Proteus vulgaris, Mycobaccerium smegmatis, Bacillus cereus, and Encerococcus faecalis adjusted
to an O.D. of 0.1 at 600 mμ. Note: For enhanced growth of M. smegmacis, add Tween 80 (1
ml/liter broth medium) and incubate for 3 to 5 days in a shaking water bath, if available.
Media
Per designated student group: seven Mueller-Hinton agar plates.
Antimicrobial Sensitivity Discs
Penicillin-G, 10 ug , streptomycin, 10 ug: tetracycline, 30 ug; chloramphenicol, 30 ug;
gentamicin, 10 ug; vancomycin, 30 ug and sulfanilamide, 300 ug.
Equipment
Sensi-disc dispensers or forceps,Bunsen burner, sterile cotton swabs, glassware marking pencil,
and millimeter ruler.
PROCEDURE
I. Place agar plates right side up in an incubator heated to 37°C for 10 to 20 minutes with the
covers adjusted so that the plates are slightly opened.
2. Label the covers of each of the plates with the name of the test organism to be inoculated.
3. Using sterile technique, inoculate all agar plates with their respective test organisms as
follows:
a. Dip a sterile cotton swab into a well-mixed saline test culture and remove excess inoculum
by pressing the saturated swab against the inner wall of the culture tube.
b. Using the swab streak the entire agar surface horizontally ,vertically and around the
outer edge of the plate to ensure a heavy growth over the entire surface.
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4. Allow all culture plates to dry for about 5 minutes.6
5. Using the Sensi-disc dispenser, apply the antibiotic discs by placing the dispenser over
the agar surface and pressing the plunger, depositing the discs simultaneously onto the
agar surface. If dispensers are not available, distribute the individual discs at equal distances with forceps-dipped in alcohol and flamed.
6. Gently press each disc down with the wooden end of a cotton swab or sterile forceps to ensure
that the discs adhere to the surface of the agar. Do not press the discs into the agar.
7. Incubate all plate cultures in an inverted position for 24 to 48 hours at 37°C.
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The clindamycin disk is used for testing susceptibility to both clindamycin and lincomycin.
Resistant strains of S .aureus ododuce b-lactamase and the testing of the 10-unit penicillin G disk
is preferred. Penicillin G should be used to test the susceptibility of all penlclllinasesensitive
penicillins. such as ampicillin, amoxacillin, azlocillin, bacampicillin, hetacillin, carbenicillin,
meziocillin, piperacillin, and ticarcillin. Results may also be applied to phenoxymethyl penicillin
or phenethicillin.
For enterococci, aerococci, and non-enterococcal streptococci, the designation "moderately
susceptible" implies the need for high dose penicillin or ampicilin for endocarcitis and serious
invasive tissue infections that may require (always for enterococci) combined therapy with an
aminoglycoside (gentamicin for improved therapeutic response and bactericidal action. Non
enterococcal streptococci should have an MIC determined 10 case of endocarditis. Urinary
isolates should be considered to be susceptible to ampicillin or penicillin alone.
Ampicillin/sulbactam, azteronam, cefotetan, ceftazidime. ceftriaxone. and imipenem are among
the most recently studied beta Iactams having a separate diagnostic disk and a generally wider
spectrum of antimicrobial aetivity. Specialy against gram-negative bacilli when compared to
previously approved cephalosporins such as cephalothin. The Cefazolin test results may not
accurately predict susceptibility to other first-generation cephalosporins. Cephalothin should be
tested instead to represent cephalothin. cefaclor (except for Haemophifus). cephapirin.
cephradine. cephalexln. and cefadroxiI. S.aureus Strains exihibting resisrance to one of the
pemcillinase-resistant penicillins (MRSA) must be reported as resistant to cephalosporins and
other newer β -lactams such as amoxicillin /clavulanic acid, Ampicillinis/ sulbactam. imipenem
and ticarcillin/clavulanic acid, regardless of in vitro rest results. This is primarily because in
most cases of documented MRSA Infection, the patient has resoonded poorly to the
cephalosporin therapy or convincing clinical data has yet to be derived confirming clinical
efficacy (clavulanic acid or sulbactam combinations and imipenem). Methicillin-resistant,
coagulase -negative Stapylococcus spp. also appears not to respond well to the above cited drugs.
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PART B: SYNERGISTIC EFFECT OF DRUG COMBINATIONS:
PURPOSE
To become acquainted with the disc-agar diffusion technique for determination of synergistic
combinations of chemotherapeutic agents.
PRINCIPLE
Combination chemotherapy, the use of two or more antimicrobial or antineoplastic agencs, is
being employed in medical practice with ever -increasing frequency. The rationale for using
drug combinations is the expectation that effective combinations might lower the incidence of
bacterial resistance, reduce host toxicity of the antimicrobial agents (because of decreased
dosage requirements), or enhance the agents' bactericidal activity. Enhanced bactericidal activity is known as synergism. Synergistic activity is evident when the sum of the effects of the
chemotherapeutic agents used in combination significantly greater than the sum of their effects
when used individually. This result is readily differentiated from an additive (indifferent)
Effect, which is evident when the interaction of two drugs produces a combined effect that is no
greater than the sum of their separately measured individual effects.
A varietv of in vitro methods are available to demonstrate synergisric activity. In this experiment a disc-agar diffusion technique will be performed to demonstrate this phenomenon. This
technique uses the Kirby-Bauer antibiotic susceptibility test procedure, as descriped in Part A of
this experiment, and requires both MuellerHinton agar plates previously seeded with the test
organisms and commercially prepared, antimicrobial-impregnated discs . The two discs, representing the drug combination, are placed on the inoculated agar plate and separated by a
distance (measured in mm) that is equal to or slightly greater than one-half the sum of their
individual.
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Zones of inhibition when obtained separately. Following the incubation period, an additive
effect is exhibited by the presence of two distinctly separate circles of inhibition. If the drug
combination is synergistic, the two inhibitory zones merge to form a "bridge" at their juncture,
as illustrated in Figure 11.3 the drug combinations to be used in this experimcntal procedure are:
1. Sulfisoxazolc, 150 μg, and trimethoprim, 5 μg. Both antimicrobial agents are enzyme
inhibitors that act scquentially in the metabolic pathway leading to folic acid synthesis. The
antimicrobial effect of each drug is enhanced when the two drugs are used in combination.
The pathway thus exemplifies synergism.
PABA
Folate synthetase
inhibited by sulfisoxazole
Dihydrofolic reductase
Dihydrotolic acid
inhibited by trimethoprim
Tetrahydrofolic acid
2. Trimethoprim 5μg, and tetracycline, 30 μg. The modes of antimicrobial activity of these two
chemotherapeutic agents differ; tetracycline acts to interfere with protein synthesis at the
ribosomes. Thus, when used in combination, these dnigs produce an additive effect.
MATERIALS
Cultures
0.85% saline suspensions of Escherichia coli and Staphylococcus aureus adjusted to anO.D. of 0.1
at 600 mμ.
Media
Per designated student group: four Mueller-Hinton agar plates.
Antimicrobial Sensitivity Discs
Tetracycline, 30 μg, trimethoprim, 5 μg; and sul- fisoxazole, 150 μg .
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Equipment
Bunsen burner, forceps, sterile, cotton swabs, millimeter ruler, and glassware marking pencil.
PROCEDURE
1. Inoculation of Mueller-Hinton agar plates: Follow steps 1 through 4 as described under the
procedure in Part A of this experiment.
2. Using the millimeter ruler, determine the center of the underside of each plate and mark
with a glassware marking pencil.
3. Using the glassware marking pencil, mark the underside of each agar plate culture at both
sides from the center mark at the distances specified below
a. E.coli--inoculated plate for trimethoprim and sulfisoxazole combination sensitivity:
12.5
mm on each side of center mark.
b. S.aureus-inoculated plate for trimethoprim and sulfisoxazole combination sensitivity 14.5 mm
on each side of center mark. .
c. E. coli and S.aureus inoculated plates for trimethoprim and tetracycline combination
sensitivity: 14.0 mm on each side of center mark.
4. Using sterile forceps, place the antimicrobial discs, in the combinations specified in step3, onto
the surface of each agar plate culture at the previously marked positions. Gently press each
disc down with the sterile forceps to ensure that it adheres to the agar surface.
5. Incubate all plate cultures in an inverted position for 24 to 48 hours at 37°C
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OBSERVATIONS AND RESULTS
Part A: Kirby-Bauer Antimicrobial Susceptibility Test Procedure
I. Examine all plate cultures for the presence or absence of a zone of inhibition surrounding each
disc.
2. Using a ruler graduated in millimeters, carefully measure each zone of inhibition to the nearest
millimeter and record your results in the chart.
3. Compare your results with Tablel1.2 and indicate in the chart the susceptibility or each test
organism to the chemotherapeutic agent as resistant (R). intermediate (1), or sensitive (S).
Refer to photo number 30 in the color-plate insert for illustration of this reaction.
Chemotherapeutic
Gram – Negative
Agent
E.coli
Zone
size
p.aeruginosa
Susceptibility
Zone
Susceptibility
size
p.vulgaris
Zone
Susceptibility
size
M. smegmatis
Zone
Susceptibility
size
Penicillin
Streptomycin
Tetracycline
Chloramphenicol
Gentamicin
Vancomycin
Sulfanilamide
Chemotherapeutic Agent
Gram Positive
S. aureus
Zone size
E. faecalis
B. cereus
Susceptibility Zone Susceptibility Zone Susceptibility
size
size
Penicillin
Streptomycin
Tetracycline
Chloramphenicol
Gentamicin
Vancomycin
Sulfanilamide
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4. for each of the chemotherapeutic agents,
indicate:
a: The spectrum of its activity as broad or limited.
b. The type or types of organisms it is effective against as gram-positive, gram-negative, or acid
fast.
Chemotherapeutic
Spectrum of Activity
Types (s) of Microorganisms
agent
Penicillin
Streptomycin
Tetracycline
Chloramphenicol
Gentamicin
Vancomycin
Sulfanilamide
Part B: Synergistic Effect of Drug Combinations
Examine all agar plate cultures to determine the zone of inhibition patterns exhibited.
Distinctly separate zones of inhibition are indicative of an additive effect, whereas a merging
of the inhibitory zones is indicative of synergism. Record your observations and results in the
chart.
Refer to photo number 29 in the color-plate insert for illustration of these
reactions.
Cultures
Appearance of zone of
Synergistic
inhibition
additive Effect
or
E. coli:
Trimethoprim
and
sulfisoxazole
trimethoprim and tetracycline
S. aureus:
Trimethoprim
and
sulfisoxazole
trimethoprim and tetracycline
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Identification of Human Streptococcal athogens
PURPOSE:
To become familiar with
1. The medical significance of streptococci.
2. Selected laboratory procedures designed to differemiate streptococci on the basis of their
hemolytic activity and biochemical patterns associated with the Lancefield group
classifications.
PRINCIPLE:
Members of the genus Streptcococcus are perhaps responsible for a greater number of infectious
diseases than any other group of microorganisms. Morphologically , they are cocci that divide in
a single plane forming chains, They form circular translucent to opaque pinpoint colonies on
solid media. All members of this group are gram -positive and many are nutritionally fastidious
requiring enriched media such as blood for growth.
The streptococci are classified by means of two major methods: (1) their hemolytic activity
and (2) the serologic classification of Lancefield.
The observed hemolytic reactions on blood agar are of the following three types:
1. (α) Alpha-hemolysis, an incomplete form of hemolysis, produces a green zone around the
colony. Alpha-hemolytic streptococci, the Screpcococcus viridans species, are usually
nonpathogenic opportunists, In some instances however, they are capable of inducing
human infections such as subacute endocarditis, which may precipitate valvular damage
and heart failure if untreated. Strepcococcus pneumoniae, the causative agent of 1obar
pneumonia, will be studied in a separate experiment.
2. (β) Beta-hemolysis, a complete destruction of red blood cells, exhibits a clear zone of
approximately two to four times the diameter of the colony. The streptococci capable of
producing beta-hemolysins are most frequemly associated with pathogenicity.
3. (γ) Gamma-hemolysis is indicative of the absence of any hemolysis around the colony, most
commonly, gamma-hemolytic streptococci are avirulent.
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Lancefield classified the streptococci into groups, designated A through 0, based on the
presence of an antigenic group-specific hapten called the C-substance. This method of classification generally implicates the members of Groups A, B, C, and D in human infectious
processes.
Beta-hemolytic streptococci belonging to Group A, and collectively referred to as Streptococcus pyogenes, are the human pathogens of prime importance. Members of this group are
the main etiological agents of human respiratory infections such as tonsillitis,
bronchopneumonia, and scarlet fever as well as skin disorders such as erysipelas and
cellulitis. In addition, these organisms are responsible for the development of complicating
infections, namely glomerulonephritis and rheumatic fever, which may surface when primary
streptococcal infections either go untreated or are not completely eradicated by antibiotics.
The beta-hemolytic streptococci found in Group B are indigenous to the vaginal mucosa and
have been shown to be responsible for puerperal fever (childbirth fever), a sometimes-fatal
neonatal meningitis, and endocarditis. Members of Group C are also beta-hemolytic and have
been implicated in erysipelas, puerperal fever, and throat infections. Group D streptococci
generally exhibit alpha- or gamma-hemolysis on blood agar plates. This group includes .theenterococci such as S. faecalis (now reclassified as Enterococcus- faecalis), which may be the
etiological agent of urinary tract infections, and the nonenterococci such as S. bovis, which is
of lesser medical significance in humans.
The virulence of the streptococci is associated with their ability to produce a wide variety
of extracellular metabolites. Included among these are the hemolysins (alpha and beta), leucocidins that destroy phagocytes, and theerythrogenic toxin responsible for the rash of scarlet
fever, Also of medical significance are the metabolites hyaluronidase (the spreading factor),
which hydrolyzes the tissue cement hyaluronic acid; streptokinase, a fibrinolysin; and the
nucleases, ribonuclease and deoxyribonuclease, which destroy viscous tissue debris. The last
three metabolic end products facilitate the spread of the organisms, thereby initiating
secondary sites of streptococcal infection.
Although the different groups or streptococci have similar colonial morphology and microscopic appearance, they can be separated and identified by the perfonnance of a variety of
laboratory tests. Toward this end, you will perfonn laboratory procedures to differentiate
among the medically significant streptococci on the basis of their Lancefield group classification
and their hemolytic patterns. Table 4.1 will aid in this separation.
Identification of Group A streptococci involves the following procedures:
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1. Bacitracin test: A filter-paper disc impregnated with 0.04 unit of bacitracin is applied to the
surface of a blood agar plate previously streaked with the organism to be identified,
Following incubation, the appearance of a zone of growth inhibition surrounding the disc is
indicative of Group A streptococci. Absence of this zone suggests a non-Group A organism.
Hemolytic activity is identified with a blood agar medium, The pathogenic streptococci, primarily the beta- hemolytic, can be separated from the generally avirulent alpha- and gamma -hemolytic streptococci by the type of hemolysis produced on blood agar as previously described.
MATERIALS:
Cultures:
24-hour blood agar slant cultures of Streptococcus pyogenes (ATCC 12385), Enterococcus faecalis
and Streptococcus viridans .
Media:
Per designated student group: three blood agar plates.
Equipment:
Bunsen burner inoculating loop, staining tray, lens paper, bibulous paper, microscope, sterile
canon swabs, glassware marking pencil
PROCEDURE:
1. Prepare a Gram stain preparation of each streptococcal culture and observe under oil
immersion. Record in the chart your observations of cell morphology and Gram reaction.
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2. Prepare the blood agar plate cultures to identify the type of hemolysis as follows:
a. With a glassware marking pencil, Write on the bottoms of blood agar plate to
accommodate test organism, Label it with the name of the culture to be inoculated.
b. Using sterile inoculating technique, make line streak of inoculation of each organism in its
respective blood plate.
3. Prepare the blood agar plate cultures for the bacitracin test as follows:
a. With a glassware marking pencil, label the cover of blood agar plate with the names of the
organisms to be inoculated S. pyogenus .
b. Using a sterile cotton swab, inoculate the agar surface of the plate with its respective test
organism by streaking first in a horizontal direction, then vertically to ensure a heavy
growth over the entire surface.
c. Using alcohol-dipped and flamed forceps apply a single O.04-unit bacitracin disc to the
surface of each plate; gently touch the disc to ensure its adherence to the agar surface.
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Identification of Streptococcus pneumoniae
PURPOSE:
To become familiar with laboratory procedures to differentiate between Streptococcus pneumoniae and other alpha-hemolytic streptococci.
PRINCIPLE:
The pneumococcus Streptococcus pneumoniae is the major etiological agent of lobar pneumonia,
an infection characterized by acute inflammation of the bronchial and alveolar membranes.
These organisms are gram-positive cocci. tapered or lancet-shaped at their edges , that occur in
pairs or as short , tight chains, The large thick capsules formed in vivo are responsible for antiphagocytic activity; which is believed to enhance the organisms' virulence. In addition the pneumococci produce alpha-hemolysis on blood agar plates. Because of these properties (short-chain
formation, alpha-hemolysis. and failure of the capsule to stain on Gram staining)
the organ-
isms closely resemble Streptococcus viridans species. The S. pneumoniae can be differemiated
from other alpha-hemolytic streptococci on the basis of the following laboratory tests:
Test
S. pneumoniae
S. mitis
Hemolysis
α
α
Bile solubility
+
-
Optochin
+
-
Inulin fermentation
+
-
Brief descriptions of the tests and their mechanisms follow:
1. Bile solubility test: In the presence of surface-active agents such as bile and bile salts
(sodium desoxycholate or sodium dodecyl sulfate), the cell wall of the pneumococcus
undergoes lysis. Other members of the alpha-hemolytic streptococci will not be lysed by
these agents and are bile-insoluble. Following incubation bile-soluble cultures will appear
clear; bile-insoluble cultures will be turbid.
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2. Optochin test: This is a growth inhibition test in which 6-mm filter-paper discs impregnated with 5 mg of ethylhydrocupreine hydrochloride (optochin) and called P-discs are
applied to the surface of a blood agar plate streaked with the test organisms. The
S.pneumoniae being sensitive to this surface-active agent, are lysed with the resultant
formation of a zone of inhibition greater than
15 mm surrounding the P-disc. Nonpneumococcal alpha-hemolytic streptococci are resistant
to optochin and fail to show a zone of inhibition or produce a zone less than 15 mm.
3. Inulin fennentation: The pneumococci are capable of fermenting inulin. while most other
alpha-hemolytic streptococci are inulin-nonfermenters. Following incubation, the acid resulting
from inulin fenT1entation will change the color of the culture from red to yellow. Cultures that
are not capable of fermenting inulin will not exhibit a color change. which is a negative test result
organisms.
MATERIALS:
Cultures:
24-hour blood agar slant cultures of Streptococcus pneumoniae and Srreptococcus viridan.
Media:
Per designated student group: One blood agar plate, two phenol red inulin broth tubes, and four
13 x .75-mm tubes containing 1 ml of nutrient broth.
Reagents:
Crystal violet, Gram's iodine, ethyl alcohol, safranin, methylene blue, 10% sodium desoxycholate, commercially available Taxo P-discs (5 mg of optochin) .
Equipment:
Bunsen burner, inoculating loop, sterile cotton swabs. sterile I-ml serological pipettes , mechanical pipetting device, 95%alcohol in beaker, forceps, and glassware marking pencil.
PROCEDURE:
I. Bile solubility test
a. Label two nutrient broth tubes S. pneumoniae and two other tubes S.viridans
b. Aseptically add 2 loopsful of the test organisms to the appropriately labeled sterile test tubes
to affect a heavy suspension.
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c. Aseptically add 0.5 ml of sodium desoxycholate to one tube of each test culture. The remaining
cultures will serve as controls.
d. Incubate the tubes in a water bath at 37°C for 1 hour.
2. Optochin test:
a. With a glassware marking pencil, divide the bottom of a blood agar plate into two equal
sections and label one section S. pneumoniae and the other S. viridans .
b. Using a sterile cotton swab, heavily inoculate the surface of each section with its respective
test organism in a horizontal and then vertical direction, being careful to stay within the
limits of each section.
c. Using an alcohol-dipped and flamed forceps, apply a single Taxo P-disc (optochin) to the
surface of the agar in each section of the inoculated plate. Touch each disc slightly to
ensure its adherence to the agar surface.
d. Incubate the plate in an inverted position for 24 to 48 hours at 37°C.
3. Inulin fennentation test:
a. Label two phenol red inulin broth tubes with the name of each test organism to be
inoculated.
b. Using sterile technique and loop inoculation, inoculate each experimental organism in its
appropriately labeled tube of medium.
c. Incubate the tube cultures for 24 to 48 hours at 37°c .
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Characterization of lactose fermenting enteric bacteria
Members of the family Enterobacteriacea are Gram –ve bacilli 3-5μ by 0.5μ .They are divided
into two groups according to their effect on lactose.
The lactose fermenters: are collectively called "Coli forms" and include E.coli, Klebsiella, and
Citrobacter.
The non lactose fermenters: Salmonella, Shigella, Proteus, Pseudomonas.
E.coli
These are normal inhabitants of intestine of man and animals however, some can cause disease in
man.
Morphology:
Gram –ve bacilli, motile.
Cultural characters:
Aerobes and facultative anaerobes, they grow on simple media.
ON Macconkey medium they give rose- pink colonies due to lactose fermentation.
Biochemical activity:
They ferment glucose, lactose, maltose, mannite, sucrose, with production of acid and gas.
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Test
E.coli
Indole test
+ve
V.P.
- ve
M.R
+ve
Citrate test
- ve
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Klebsiella
27 serotype according to capsular polysaccharide.
Morphology:
Gram – ve bacilli, non motile, and capsulated.
Cultural characters:
Pink colonies on Macconkey colonies are mucoid due to extra cellular slime in abundant amount.
Test
Klebsiella
Indole test
-ve
V.P.
+ ve
M.R.
- ve
Citrate test + ve
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Carbohydrate Fermentation:
Glucose fermentation:
Depends on the varying ability of bacteria to ferment sugars with acid production which may or
may not evolve gas.
Experiment: growing the organism in fluid medium composes of peptone water to which a
sugar is added in 1% conc.
Results:
Pink if acid is produced +ve result.
Yellow -ve result.
Indole test:
Demonstrate the ability of certain bacteria to decompose the amino acid tryptophane present in
peptone to Indole.
Experiment:
1-
Inoculate the organism in peptone water and after incubation for 24 h. At 37ْc .
2-
Kovac´s reagent or Ehrlich´s reagent is added.
3-
If pink ring is produced the organism is Indole +ve.
4-
If yellow ring is produced it is Indole – ve.
MRVP:
Vogues- Proskaur´s reaction:
Some bacteria ferment glucose with production of acetyl methyl carbinol.
Exp.:
Growing bacteria on glucose phosphate peptone then concentrated potassium hydroxide is added
if an eosin –pink colour is produced it means positive results.
Methyl red test (MR):
This test is done to detect the ability of some bacteria to produce large amounts of acid on
fermentation of glucose thus lowering the PH of the medium below 4.
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Exp:
Growing the organism on glucose phosphate peptone .
Incubate at 37 ْc for 48h. .
Few drops of the Methyl red indicator are added.
+ve test → bright red colour.
- ve test → yellow colour.
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Characterization of non lactose –fermenting organisms
Proteus
Are found in stools of man and animals, they cause wound infection, otitis media&UTI.
Two important species are Proteus vulgaris &Proteus mirabilis.
Morphology:
Gram –ve bacilli, very pleomorphic, highly motile, and non capsulated.
Cultural characters:
Nutrient agar: they give colonies which swarm in successive waves.
Macconkey:
Compact pale non –lactose fermenting colonies are formed.
Biochemical activities:
Urease test
+VE
Ferment glucose VE+
Indole test
VE+
M.R.
VE+
V.P.
VE-
Pseudomans
Ps. Aeruginosa is the commonest human pathogen of the pseudomonas group. It is found in soil,
sewage some are commensals others cause UTI wound infection and otitis media.
The organism is highly resistant to antibiotics and cause chronic infection in low immune person.
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Morphology:
Gram –ve bacilli, motile non capsulated and non sporing.
Nutrient agar: aerobes leading to greenish discolouration of the medium due to its diffuse
exopigment pyocyanin and fluoricin.
Mac conkey:pale non-lactose fermenting colonies are formed.
Biochemical activities:
Ferment glucose -VE
Oxidase test
VE+
VP
VE -
MR
VE-
Urease test:
Some organisms e.g. Proteus produce urease enzyme which split urea with the release of
ammonia it causes alkalinity and increase pH of the surrounding medium.
Expirement:
Growing the organism on a medium containing urea and phenol red indicator urease +ve
organisms will turn the medium deep pink after 24 hours.
Oxidase test:
Some bacteria e.g. Neisseria,Vibrio,Pseudomonas produce oxidase enzyme.the later can reduce the
oxidase reagent (tetra methyl-p-phenylene- diamine hydrochloride) to a deep purple colour.
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Expirement:
The test is done by picking up a portion of the colony tested and smearing it on a strip filter
paper impregnated with oxidase reagent (1% freshly prepared) the immediate development of a
deep purple colour indicate a + ve test.
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Characterization of Salmonella&Shigella
There are more than 2000 serotypes within genus Salmonella, four of them S.typhi, S.paratyphi A
,B , S, C, cause enteric fever in man other species cause Salmonella food poisoning in man.
Morphology:
Gram –ve bacilli, motile, non capsulated.
Cultural characters:
Aerobes and facultative anaerobes grow on nutrient agar and Macconkey produce pale non –
lactose fermenting colonies.
Biochemical activities:
The triple sugar iron (TSI) agar test:
Used to differentiate between Entero bacteriaceae on the basis of carbohydrate fermentation and
hydrogen sulphide production.
TSI agar slants: 1) Contain lactose and sucrose 1% conc.
2) Acid base indicator phenol red which changes from orange to yellow in presence of acids.
3) The slant is inoculated by means of a stab- and streak procedure.
Result with Salmonella:
Alkaline slant (red)
Acid butt (yellow)
H2S produce black colour.
Only glucose fermentation has occurred acid produced on the slant surface is oxidized rapidly. In
the butt the acid reaction is maintained because of reduced O2 tension and slower growth of
organism.
H2S production is determined by Sodium thiosulfate which is added to the medium determined
by blacking in the butt. Because of precipitation of insoluble ferrous sulphide.
Shigella
Morphology:
Gram –ve bacilli non motile non capsulated.
Cultural characters:
They produce pale non- lactose fermenting colonies on Macconkey
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Biochemical activities:
TSI: alkaline (red) &acid butt (yellow) no H2S.
Only glucose fermentation has occurred.
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Identification of Enteric micro –organisms using Computer –Assisted Multi test
Micro system API -20
Several multitest systems have been developed for differentian and identification of members of
the Enterobacteriaceae they use micro techniques that incorporate a number of media in single
unit.
It employs plastic strip Composed of 10&20&32 individual micro tubes, each contain
dehydrated medium in the bottom and an upper cupule.
The media become hydrated during inoculation of a suspension of the test organism .The strip
is then incubated in a plastic covered tray to prevent evaporation.Following incubation
identification of the organism is made by using differential charts supplied by the
manufacturer.
We must noticed that underlined test is anaerobic must be covered by sterile oil.
Test surrounded by U the cupule must be full above the level. Certain test ex. Need special
reagents e.g. INDOLE, VP. Following incubation you will make your identification by:
1- The traditional method of noting characteristic color changes and interpreting them
according to manufacturer instructions.
a-The 21 test are divided into seven groups of three each.
b-A value of 1 is assigned to the 1 st+ve test in each group.
c-A value of 2 is assigned tu the 2nd +ve test in each group.
d-A value of4 is assigned to the 3rd +ve test in each.
E-A 7-Digit tulmber is obtained by totaling the +ve values of each of the seven groups of three
this number is located in the analytical profile index to identify the organism.
2- The computer assisted method called (mini API).
MiniAPI:
Theory:
I-Spectrophotometric.
2-Nephlometric.
3- Turbidmetry
They are colorimetric methods. It is used for
assesment of:
I-Certain enzyme reaction e.g. urease.
2-Break down of certain intermediate e.g. Indole.
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3- Turbidity: ability to grow in certain medium e.g. Citrate. 4-Substrate changes e.g. sugar
break down& acid production. 5-Biosynthesis of certain intermediates such as metabolism of
nitrate. Data handling in this machine depend on:
Coloremetric+ Mathematical models.
Advantatge:
I-Minimal storage space.
2- The use of less media.
3-Minimize time to obtain results.
4-The applicability of results to a computerized system for identification of
organism.
Disadvantatge:
I-Difficulty in obtained a standardized inoculum.
2-Presence of air bubbles during insertion of the sample.
PURPOSES:
1. To become familiar with the members of, the
family Enterobacteriaceae.
2. To become familiar with laboratory procedures designed to identify enteric pathogens
using commercial multi test microsystems.
INTRODUCTION:
The Enterobacteriaceae are a significant group of bacteria that are endogenous to the intestinal
tract or that may gain access to this site via a host's ingestion of contaminated food and water.
The family consists of a number of genera whose members vary in their capacity to produce disease. The Salmonella and Shigella are considered to be pathogenic. Members of other genera,
particularly Escherichia and Enterobacter and to a lesser extent Klebsiella and Proteus, constitute
the natural flora of the intestines and are generally considered to be avirulent.
Remember, however, that all can produce disease under appropriate conditions.
The Enterobacteriaceae are gram-negative, short rods. They are mesophilic, nonfastidious
organisms that multiply in many foods and water sources. They are all non-spore-formers and
susceptible to destruction by common physical and chemical agents.
They are resistant to destruction by low temperatures and Can therefore frequently survive in
soil, sewage, water, and many foods for extended periods of time.From a medical point of view,
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the pathogenic Emerobacteriaceae are salmonellae and shigellae. Salmonellae are responsible for
enteric fevers, typhoid and milder paratyphoid and gastroenteritis. In typhoid Salmonella typhi
penetrates the intestinal mucosa and enters the blood stream thus infecting organs such as the
gall bladder, intestines, liver, kidney, spleen, and heart. Ulceration of the intestinal wall caused
by the release of the lipopolysaccharide endotoxin into the blood over a long febrile period, and
en-teric symptoms are common. Gastroenteritis is caused by a number of Samonella species.
Symptoms associated wirh this type of food poisoning include abdominal pain, nausea, vomiting,
and diarrhea which develop within 24 hours of ingestion of contaminated food and last for
several days.
Several Shigellae are responsible for shigellosis, bacillary dysentery that varies in severity,
Ulceration of the large iniestine explosive diarrhea, fever, and dehydration occur in the more
severe cases.
Isolation and identification of enteric bacteria from feces, urine, blood and fecally
contaminated materials are of major importance in diagnosis of enteric infections. Although the
Enterobacteriaceae are morphologically a like and in many ways metabolically similar, laboratory procedures for the identification of these bacteria are based on differences in biochemical
activities.
In the past, several multiest systems have been developed for differentiation and identification of members of the Enterobacteriaceae. They use microtechniques that incorporate a
number of media in a single unit. At least six multi test systems are commercially available. The
obvious advantages of these units are the need for minimal storage space, the use of less media,
the rapidity with which results may be obtainedand the applicability of the results to a
computerized system for identification of oganisms, There are also certain disadvantages with
these systems,including difficulty in obtaining the proper inoculum size since some media
require heavy inoculation while others need to be lightly inoculated, the possioility of media
carryover from one compartment to another, and the possibility of using inoculum of improper
age. Despite these difficulties, when properly correlated with other properties such as Gram
srain and colonial morphology on specialized solid media, these systems are acceptable for the
identification of Enterobacteriaceae. The most frequently used systems are discussed.
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ENTEROTUBE MULTITEST SYSTEM AND ENCISE ІІ :
The Enterotube Multitest System (Roche Diagnostics, Hoffmann-La Roche, Inc., Nutley, New
Jersey) consists of a single tube containing 12 compartments (Figure 8.2) and a self enclosed
inoculating needle. This needle can touch a single isolated colony and then in one operation be
drawn through all 12 compartments, thereby inoculating all of the test media. In this manner, 15
standard biochemical tests can be performed in one inoculating procedure. Following incubation,
the color changes that occur in each of the compartments are interpreted according to the
manufacturer's instructions to identify the organisms. This method has been further refined to
permit identification of the enteric bacteria by means of a computer assisted System called
ENCISE (Enterobacteriaceae numerical coding and identification system for enterotube).
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API (ANALYTICAL PROFILE INDEX) SYSTEM
The API-20E (Analytab Products 1nc. Plainview,New York) Employs a plastic strip composed of
10 individual microtubes, each containing a dehvdrated medium in the bottom and an upper
cupule as shown in Figure 8.3 . The media become hydrated during inoculation of a suspension
of the test organism, and the strip is then incubated in a plastic-covered tray to prevent
evaporation. In this manner 22 biochemical tests are performed. Following incubation,
identification of the organism is made by using differential charts supplied by the manufacturer
or by means of a computer-assisted system called PRS (profile recognition system).
PRS includes an API coder profile register, and selector.
In the following experiment you will inoculate an Enterotube and an API strip with an
unknown enteric organism. Following incubation, you wiII make your identification by two
methods:
(1) The traditional method of noting the characteristic color changes and interpreting them
according to manufacturers' instructions.
(2) The computer-assisted method illustrated in Figure 8.4.
MATERIALS:
Cultures:
Number- coded. 24-hour trypticase soy agar streak plates of Escherichia coli, Salmonella
typhimurium, Klebsiella pneumoniae, Enterobacter aerogenes, Shigella dysenteriae, and Proteus
vulgaris.
Media:
Per designated student group: One Enterotube ІІ one API-20E strip, and one 5-ml tube of 0.85%
sterile saline.
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Reagents:
Sterile mineral oil 10% ferric chloride, Kovac's reagent, V-P reagent for API sestem, nitrate
reduction reagents, Barritt's reagent (V-P test reagent for Enterotube system), 1.5% hydrogen
peroxide, and 1 % p-aminodimethylanaline oxalate (oxidase reagent).
Equipment:
Bunsen burner, inoculating loop, 5-ml pipette, mechanical pipetting device, sterile Pasteur
pipettes, glassware marking pencil, API profile recognition system and differential identification
charts, and Enterotube ENClSE pads and color reaction charts.
PROCEDURE:
API-20E System
1. Familiarize yourself with the components of the system: Incubation tray, lid, and the strip
with its 20 microtubes.
2. Label the elongated flap on-the incubation tray with your name and the number of the
unknown culture supplied by the instructor.
3. With a pipette, add approximately 5 ml of tap
water to the incubation tray.
4. Using a sterilized loop, touch an isolated colony on the provided streak-plate culture, transfer
the inoculum to a 5-ml tube of sterile saline, and mix well to effect a uniform suspension.
5. Remove the API strip from its sterile envelope and place it in the incubation tray.
6. Tilt the incubation tray. Using a sterile Pasteur pipette containing the bacterial saline
suspension, fill the tube section of each compartment by placing the tip of the pipette against
the side of the cupule: Fill the cupules in the CIT, VP, and GEL microtubes with the
bacterial suspension.
7. Using a sterile Pasteur pipette, fill the cupules of the ADH, LDC, ODC, and URE microtubes
with sterile mineral oil to provide an anaerobic environment.
8. Cover the inoculated strip with the tray lid and incubate for 13 to 24 hours at 37°C.
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Enterotube System:
1. Familiarize yourself with the components of the system: Screw caps at both ends, mediumcontaining compartments, self enclosed inoculating needle, plastic side bar, and blue taped
section.
2. Label the Entrerotube with your name and the number of the
unknown culture supplied by the instructor.
3. Remove the screw caps from both ends of the Enterotube. Using the inocubting needle
contained in the Enterotube, aseptically pick some inoculum from an isolated colony on the
provided streak-plate culture.
4. Inoculate the Enterotube as follows:
a. Twist the needle in a rotary motion and withdraw it slowly through all 12 compartments.
b. Replace the needle in the tube and with a rotary motion push the needle into the first three
companments (GLU, LYS, and ORN). The point of the needle should be visible in the
H2S/IND compartment.
c. Break the needle at the exposed notch by bending, discard the needle remnant, and replace
the caps at both ends. The presence of the needle in the three compartments maintains
anaerobiosis, which is necessary for dextrose fermentation, Co2 production, and the
decarboxylation of lysine and ornithine.
J. Remove the blue tape covering the ADO, LAC, ARB, SOR, VP, DUL/PA, URE, and CIT
compartments. Beneath this tape are tiny air vents that provide aerobic conditions in these
compartmems.
6. Place the clear plastic slide band over the GLU compartment to contain the wax, which may
be spilled by the excessive gas production of some organisms.
7. Incubate the tube on a flat surface for 24 hours at 37°C.
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Table 8.1 Summary of Results: 18-24 Hour Procedure API 20E) System'
Interpretation of Reactions
Tube
Positive
Yellow
ONPG
ADH
LOC
ODC
Negative
Incubation
18-24 hours
36-48 hours
Colorless
Red to orange
Yellow
Red to orange
Red to orange
Yellow
36-48 hours
Red to orange
Yellow
18-24 hours
Red to orange
Yellow
36-48 hours
Red to orange
Turquoise to
dark blue
Yellow
Light green to
yellow
Black
Red to orange
URE
No black
deposil
deposit
H,S
18-24 hours
36-48 hours
Red
orange
Any shade of orange within 18-24 hours is a
positive reaction. At 36-48 hours, orange
decarboxylase
reactions
should
be
interpreted as negative.
Orange reactions occurring at 36-48 hours
should be interpreted as negative.
(1) Both the tube and cupule should be filled.
(2) Reaction is read in the aerobic (cupule)
area.
(1) H,S production may range from a heavy
black deposit to a very thin black line around
the tube bottom. Carefully examine the
bottom of lhe tube before considering the
reaction negalive.
(2) A "browning" of the medium is a negative
reaction unless a black deposit is present.
"Browning" occurs wilh TDA-positive
organisms.
Yellow
Yellow to
TDA
Add 1 drop of 10% ferric chloride.
Brown.red
Yellow
IND
Add 1 drop 0f Kovacs' reagent.
Red ring
Yellow
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(I) Any shade 0f yellow is a posilive reaction.
(2) VP tube, before the addilion of reagents,
can be used as a negative conlrol.
Orange reactions occurring at 36-48 hours
should be interpreted as negative.
Yellow
18-24 hours
CIT
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A method 01 lower sensitivily has been
chosen. Klebsiella. Proteus and Yersinia.
routinely give iJosilive reacticns.
(1) Immediate reaction. (2) Indole positive
organisms may produce a golden-orange
color due 10 indole production. This is a
negative reaction.
(1) The reaction should be read within 2
minutes after the addition 0f the Kovacs'
reagent and the results recorded.
(2) After several minules, the HCI present in
Kovacs' reagent may react with the plastic 0f
the cupule resulting in a change from a
negative (yellow) color to a brownish red.
This is a negative reaction.
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Table 8.1 Continued
Interpretation of Reactions
Tube Posltive
VP
GEL
GLU
Negative
Comments
Add 1 drop 0f 40% polassium
(1) Wait 10 minutes belore considering
the reaction negative.
hydroxide,then 1 drop of 6% alpha.
(2) A pale pink color (after 10 minutes)
naphthol
should be interpreted as
negative. A pale pink color that appears
Red
Colorless
immediately after the
addition of reagents but turns dark pink or
red after 10 minutes
should be interpreted as positive.
Motility may be observed by hangingdrop or wet mount
preparation.
(1) The solid gelatin particles may
Diffusion of the
No diffusion
spread throughout the tube
pigment
after inoculation. Unless di/lusion
occurs, the reaction is
negative.
(2) Any degree 0f dilfusion is a
positive reaction.
Blue to blue- C
Yellow gray
Fermentation (Enterobacteriaceae,
O
green
M
M
E
N
T
S
F
O
R
Aeromonas, Vibrio)
(1) Fermentation of the
carbohydrates begins in the most
anaerobic portion (bollom) 01 the
tube. Therefore, these
reactions should be read from the
bollom of the tube to the
lop. (2) A yellow col or al the
botlom of Ihe lube indicates a
weak or delayed positive reaclion.
A
MAN
INO
Yellow
Blue
to
blue-
green
L
L
C
SOR
A
RHA
R
B
SAC
O
MEL
H
Y
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Oxidation (other gram-negatives)
. (1) Oxidalive utilization of the
carbohydrates begins in the
most aerobic portion (top) of the
tube. Therefore. these
reactions should be read from the
top to the botlom 01 the
tube. (2) A yellow color in the upper
portion of the tube and a
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D
AMY
R
ARA
A
T
E
S
GLU
NITRATE
REDUCTION
After reading GLU reaclion, add 2
drops of 0.8% sul/anilic acid and 2
drops 01 0.5% N, N-dlmelhyl.alpha
naphlhylamine
NO2
Red
Yellow
N2 gas
Bubbles : yellow
Orange after
After
reagents
reagents and
And zinc zinc
MAN
INO. or
After reading carbohydrate reaction,
add 1 drop of 1.5% H2O,.
SOR fOR
Bubbles
Calalase
bubbles
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blue in the bottom of the tube
indicates oxidative utilization of
the sugar. This reaction should be
considered positive only
for
non.Enterobacteriaceae
gram.negative rods. This is a
negative reaction for fermentative
organisms such as
Enterobacteriaceae.
(1) Belore addition of reagenls, observe
GLU t\.lbe (positive or
negative) for bubbles. Bubbles are
Indir-a!ive of reduction or
nitrate 10 the nitrogenous (N2) slat A
pOSitive reaction may
take 2-3 minutes for Ihe red color 10
appear. (3) Confirm a
negalive lest by adding zinc dust 01 20
mesh granular zinc. A
pink to orange color aller 10 minules
confirms a negative
reaction. A yellow color indicates
reduction 01 nitrates to the
nitrogenous (N,) slate.
(1) Bubbles may take 1-2 minut.es to
appear (2) Best results will
be obtained if the lest is run in tubes
thal have no gas from
lermentation.
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Table 8.2 Summary of Chemical and Physical Principles of the Tests on the API 20
E System:
Tube
ChemIcal/Physical Principles
Reactive Ingredients
ONPG
Hydrolysis 0F ONPG by beta.galactosidase releases yellow orthonitrophenol from the colorless
ONPG; IPTG (isopropylthio- galactopyranoside) is used as the inducer.
ONPG
IPTG
AOH
Arginine dihydrolase transforms arginine into ornithine,ammonia, and carbon dioxide. This causes
a pH rise in the aci-.buffered system and a change in the indicalor from yellow to red.
Arginine
LOC
Lysine decarboxylase transforms lysine into a basic primary amine, cadaverine. This amine
causes a pH rise in the acid, buffered system and a change in the indicator from yellow to red.
Lysine
OOC
Ornithine decarboxylase transforms ornithine into a basic primary amine, putrescine. This amine
causes a pH rise in the acid,buffered system and a change in the indicator from yellow to red.
Ornithine
CiT
-
Carate is the sole carbon source. Citrate utilization resulls in a pH rise and a change in the indicator
from green to blue.
Sodium cltrate
H2S
Hydrogen sulfide is produced from thiosuffale. The hydrogen sulfide reacts with iron salts to
produce a black precipitate.
SodiumThiosulfale
URE
Urease releases ammonia From urea; ammonia causes the pH to rise and changes the indicator from
yellow to red.
Urea
TDA
Tryptophan deaminase forms indolepyruvic acid from tryptophan. Indole pyruvic acid produces a
brownish-red calor in the presence of ferric chloride.
Tryptophan
IND
Metabolism 0F tryptophan results in the formation of indole. , Kovacs' reagent forms a colored
complex (pink to red) with indole.
Tryptophan
VP
Acetoin, an intermediary glucose melabolile, is produced from sodium pyruvate and indicated by the
formation of a colored complex. Conventional VP tesls may take up 104 days, but by using sodium
pyruvate, API has shortened the required test lime. Creatine intensifies the color when lests are
positive.
Sodium pyruvale,
Creatine
GEL
Liquefaction of gelatin by proteolytic enzymes releases a black pigment that diffuses throughout the
tube.
. Kahn charcoal,
gelatin
GLU
MAN
INO
SOR
RHA
SAC
MEL
AMY
ARA
Utilization of the carbohydrate results in acid formation and a consequent pH drop. The indicator
changes from blue to yellow.
GLU
Nitrate
reduction
Nitrites form a red complex with sulfanilic acid and N, N-dimethyl- alpha-naphthylamine. In case of
a negative reaction, addition 01 zinc confirms the presence of unreduced' nitrates by reducing them
to nitrites (pink to orange calor). If Ihere is no calor change after the addition of zinc, this is
indicalive of the complete reduction of nitrates through nil riles 10 nitrogen gas or 10 ananaerogenic
amine.
MAN,
INO, or
SOR for
Catalase
Catalase releasEs oxygen gas from hydrogen peroxide.
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Glucose
Mannitol
Inositol
Sorbitol
Rhamnose
Sucrose
Melibiose
Amygdalin
Arabinose
Potassium nitrale
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OBSERVATIONS AND RESULTS API-20E System:
1. Observe all reactions in the API strip that do not require addition of a test reagent, and
interpret your observations using the manufacturer's instructions. Record your observations
and results in the chart.
2. Add the required test reagents in the following order: Kovac's reagent to IND, VP reagent
to VP (read the result after 15 minutes), ferric chloride to TDA. nitrate reagents to GLU,
and oxidase reagent to OXI. Note color changes and interpret your observations according
to the manufacturer's instructions.
Record your observations and results in the chart.
3. Based on your results, identify your unknown organism using the differential identification
chart.
4. Determine and record in the chart the seven-digit profile number as described in Figure
65.4.
Identify your unknown organism by referring to the profile recognition system.
Enterotube II System :
1. Observe all reactions in the Enterotube except IND and VP, and interpret
your observations using the manufacturer's instructions. Record your
obsenvations and results in the chart.
2. Perform the IND and VP tests as follows:
a. Place the Enterotube in a rack with the GLU and VP compartments facing
downward.
b. With a needk and a syringe, gently pierce the plastic film of the H2S/IND
compartment and add2 or 3 drops of Kovac's reagent. Read the results after 1 minute.
c. As in step 2b, add 2 drops of Barritt's reagent to the VP compartment and
read
the results after 20 minutes.
d. Record your IND and VP observations and results.
3. Based on your results, identify your unknown organism using the manufacturer's color
identifica tion charts.
4. Determine and record in the chart the five-digit ID value as described in Figure 65.4,
Identify your unknown organism by referring to the computer coding manual.
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Ap1-20E
Appearance
(color)
Code
Name
OPNG
Beta-galactosidase
AHD
Arginine dihydrolase
LDc/LYS
Lysine decarboxylase
ODC/ORN
Enterotube
Result
(+) or (-)
Appearance
(color)
Result
(+) or (-)
Orinithine
decarboxylase
ClT
Citrate
H2S
Hydrogen sulfide
URE
Urease
Tryptoptl_an
TDA
deaminase
IND
Indole
VP
Acetonin
GEL
Gelatin
GLU
Glucose
MAN
Mannitol
INO
Inositol
SOR
Sorbitol
RHA
Rhamnose
SAC
Sucrose
ME
Melibiose
AMY
Amygdalin.
ARAlARB
Arabinose
OXI
Oxidase
ADO
Adonitol fermentation
GAS
Gas production
PHElPA
Phenylalanine
LAC
Lactose
DUL
Dulcitol
Organism
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Demonstration of the following items:
1-Mycobacterium tuberculosis slide: by Ziel –Neelsen stain thin pink rods singly or in small
groups.
2-Clostridium titani: Gram +ve bacilli swollen at one end due to terminal spherical projecting
spores (drum stick appearance).
3-Loffler´s serum: It is enriched media useful for growth of Diphtheria bacilli it is composed of
three parts sterile ox sheep or horse serum and one part glucose broth the medium is
rendered solid by heating it in the inspissator at 80 ْc for 2 hrs. This leads to coagulation of the
serum the medium is opaque white.
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4-Lowenstien Jensen medium (L.J.):
Selective medium used for cultivation of Tubercle- bacilli. The medium is mainly composed of
beaten egg, mineral salts and malachite green. the later inhibits the growth of bacteria other
than T.B. the medium is rendered solid by heating it in the inspissator at 80ْ c for 1 h .it is
opaque green in colour.
5-Cooked meat broth medium: This is an anaerobic culture medium which consists of minced
meat to which broth is added the medium is sterilized in the autoclave. The meat contains
reducing substances e.g. .haematin and glutathione .which maintain anaerobic conditions at
the depth of fluid medium it is suitable for clostridium group.
6-Anaerobic cultivation:
Anaerobic cultivation is essential for some bacteria which cannot grow in presence of O2e.g.
Clostridia, anaerobic streptococci, actinomyces, bacteroids,…
Anaerobiosis can be achieved either by removal of O2 by combining it with hydrogen to form
water in the presence of a palladium catalyst or by the addition of reducing substances to
culture media.
Anaerobic gas packs system:
An especial jar with a tightly fitting lid is used. Suspended from the lid is a cold catalyst which
consists of alumina pellets coated with palladium and contained a gauze sachet. In this system
hydrogen is generated inside the jar by placing a special gas pack envelops to which 10 ml of
water is added it will release Co2 and hydrogen.
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Campylobacter:
C. jejuni and C.coli are now among the commonest cause of enterocolitis especially in children.
Morphology: Small Gram negative rods comma or S-shaped. Motility is of the darting type
with cork screw like movement.
Laboratory diagnosis:
Cultural characters:
In case of diarrhea, a stool sample is inoculated on blood agar containing antibiotics
Skirrow´s medium containing; vancomycin, polymyxin and trimethoprim is a selective
medium used for their isolation from stools.
Campylobacter are microaerophilic; grow best in presence of 5% oxygen and 10%CO2. Better
growth is obtained at 42ْcْ .they are non haemolytic on blood agar.
Biochemical reactions:
They are oxidase and catalase positive but urease negative non –proteolytic and unable to
attack carbohydrates.
Haemophilus-influenzae
This organism is often found in normal throats, however it may cause meningitis and
epiglotitis in children and it also causes respiratory disease complications (bronchitis,
pneumonia, otitis media, and sinusitis) in patients with viral Influenza.
Morphology:
Gram –ve small cocco bacilli 1.5 u by 0.5u long filamentous forms occur.
Cultural characters:
Aerobes require for their growth blood containing media ,blood agar better chocolate agar to
provide haematin (x factor) and diphosphopyridine nucleotide (v factor) colonies are small
and transparent.
Neisseria
Many of members in the genus Neisseria occur as commensals in the mouth , throat , pharynx
and vagina.
Two members N.mengitidis and N.gonorrhoeae occur intracellular in pus cells.
Morphology:
Gram –ve diplococci intracellular is diagnostic and extra cellular.
Cultural characters:
Neisseria are fastidious and require blood for growth as in blood agar or chocolate agar
Theyer-martin is a selective medium used for isolation of pathogenic Neisseria.
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Identifecation Scheme of Isolated unknown Bacteria
The main lines of identification are: when you handled the sample start by doing culture keep
in the incubator then take smear.
1-Culturel appearance:
This includes the colony morphology size, shape, margin, elevation, colour, opaque or
translucent, mucoid or dry, pigment producing.
2-Microscopic examination:
a- Examination of unstained preparation will help in demonstrating motility.
b- Examination of Gram stained preparations: will determine the staininig reaction of the
organism whether Gram +ve or Gram –ve, their morphology (cocci, bacilli) size and
arrangement.
3-Biochemical reactions:
a- Sugar fermentation
b- IMVC
C- Urease test
d- Catalase test
e- Oxidase test
f- API-20 system
4- Serological identification
5- Animal inoculation
6- Bacteriophage typing:
This used for identification and typing of several species of bacteria e.g Staphylococci.
7- Molecular identification and typing methods:
These involve detection of microbial nucleic acid by PCR.
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Species Identification of, Unknown Bacterial Cultures
PURPOSE:
To identify an unknown bacterial species by the use of dichotomous keys and Bergey´s
.Manual of Systematic Bacteriology.
PRINCIPLE:
At this point in the course, you have developed the manipulative skills and the cognitive
microbiological knowledge to Identify microorganisms beyond their genus classification to
the Ievel of their species identification. Therefore in this experiment, you will use
dichotomous keys. Bergey's Marwal of Systematic Bactcriology, and information accrued
from prcviously performed laboratory procedures to help identify the species of an unknown
culture.
In Experiment 31. "Genus Identification of Unknown Bacterial Cultures," you were
required to use a varietv of biochemical tests to successfully accomplish the experimental
purpose; Your review of the required procedures and ensuing results should indicate that
only a few of these tests were actually necessary, in most instances, for the identification of
the unknown culture. Similarly, species identification can be accomplished by using a limited
number of carefully selected laboralory procedures. Notice that what appears to be a
spurious result in some cases, one that departs from the expected norm for a particular
species, may be attributable to strain differences within the given species. These
nonconforming results may be verified by the use of Bergey´s Manual to ascertain the
existence of variable biochemical test results for the particular species being studied.
In this experimental procedure, you will receive a mixed culture containing a gram positive and a gram-negative organism. The protocol will require:
(1) Gram staining.
(2) streak-plating for observation of colonial characteristics.
(3) Use of selective media for the preparation of pure culture.
(4) The performance of appropriate biochemical tests s indicated in the dichotomous keys
outlined in Figures 15.1 and 15.2 and(5) information in Bergey's Manual.
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MATERIALS:
Cultures:
Per student: Number-coded , 24- to 48 – hour mixed trypticase soy broth cu1ltures each
containing a gram-positive and a gram-negative organism selected from the species listed in
figures 15.1 and 15.2
Media:
Per Student One trypticase soy agar plate, two trypticas soy agar slants, one trypticase soy
broth, one phenylethyl alcohol agar plate, and one MacConkey's agar plat.
Required media for the biochemical tests listcd in Figures15.land 15.2should be available
on your request.
Reagents:
Crystal violet, Gram's iodine. 95% ethyl alcohol. safranin. and required reagents for the
interpretation of the biochemical reactions listed in Figures 15.l and 15.2
Equipment:
Bunsen burner, inoculating loop and needle, staining tray, immersion oil , lens paper,
bibulous paper. microscope, and glassware marking pencil.
PROCEDURE:
Session 1: Separation of the Bacteria in Mixed Unknown Culture:
1. Prepare a trypticase soy agar broth subculture of the unknown and refrigerate following
incubation. You will use this culture if contamination of the test culture is suspected
during the identification procedure.
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2. Prepare a Gram-stained smear of the original unknown culture.
3. Prepare four-way streak inoculations on the following media for the separation of the
microorganisms in the mixed cultures:
a. Trypticase soy agar for observation of colonial characteristics.
b. Phenyl ethyl alcohol agar for isolation of gram-positive bacteria.
c. MacConke‫׳‬s agar for isolation of gram negative bacteria.
4. Incubate all the plates in an inverted position and the subculture for 24 to 48 hours at
37°C.
Session 2: Preparation of Pure Cultures:
I. Isolate a discrete colony on both the phenylethyl alcohol agar plate and the MacConkey's
agar plate and aseptically transfer each onto a trypticase soy agar slant .Incubate the
trypticase soy agar slants for 24 to 48 hours at 37°C.
Session 3: Identification of Unknown Bacterial Species :
1. Prepare a Gram-stained smear from each of the trypticase soy agar slant cultures to verify
their purity by means of the Gram reaction and cellular morphology.
2. If each Gram-stained preparation is not solely gram-positive or gram-negative, repeat the
steps in Sessions 1 and 2 using the refrigerated trypticase soy agar subculture as the test
culture.
3. If the isolates are deemed to be pure on the basis of their cultural and cellular morphologies, continue with the identification procedure. During this period and in subsequent
sessions, use the dichotomous keys in Figures 15.1 and L5.2to select and perfonn the necQatar University
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essary biochemical tests on each of your isolates for identification of their species.
Incubate all cultures for 24 to 48 hours at 37°C prior to making your observations.
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Name --------------------------------Lab section -------------------------Experiment 12 species Identification of Unknown Bacterial Cultures
---------------------------------------------------------------------------------------------------------Report Sheet
Unknown Tube No. -----------------------Unknown # 1
Unknown # 2
Gram reaction and shope: ---------------------
-----------------------
Results on BAP: -----------------------------------
-----------------------
Results on MacConkey / Emb: ----------------
-----------------------
Unknown # 1
Unknown # 2
Tests performed
Results
Tests performed
Results
Conclusions :-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Unknowns are:
#1 ----------------------------#2 -----------------------------
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MYCOLOGY
There are thousand species of fungi. Most of them are saprophytes. Few species cause disease
in man or animals. Fungi can be classified morphologically or clinically.
1-Morphological classification:
A-Yeasts: These are oval or round cells that reproduce by budding e.g.Candida and
Cryptococcus neoformans.
b- Filamentous fungi: These are branching filaments (hyphae) which may be septate or
non-septate e.g. the dermatophytes ;( Microsporum, Tricophyton and Epidermophyton) and
Aspergillus.
c-Dimorphic fungi: These occur in 2 forms ; a yeast form in tissues or when grown at 37ْc;
and a filamentous from when grown at 22ْc,e.g.Histoplasma, Blastomyces, Coccidioides.
2- Clinical classification:
a-The superficial mycoses: These are fungal infections that are confined to the stratum
corneum without tissue invasion e.g. tinea versicolor (Malassezia furfur).
b -Cutanneous mycoses: These are fungal infections that involve the skin, nail or hair with
tissue destruction and immunological reaction e.g. dermatophytes and cutaneous candidiasis.
c- The subcutaneous mycoses: These are infections confined to the subcutaneous tissue
without dissemination to distant sites e.g.mycetoma.
d- Systemic mycoses: These are primary pulmonary lesions that may disseminate to any
organ e.g. dimorphic fungi. They also include a group of opportunistic mycoses e.g. systemic
candidiasis and cryptococcosis.
Fungi may also be involved in allergic conditions due to aspergillus spores.
Mycoses: fungal infection which lead to diseases.
Mycotoxicosis: fungi may elaborate toxic substances e.g. afla toxin from Aspergillus flavus
which is carcinogenic and hepatotoxic.
Candida albicans
C. albicans , is the most important species of Candida. Other species include
C.tropicalis,C.parapsilosis, C.krusei.
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C.albicans are gram positive oval budding yeast which produce pseudohyphae.It is part of the
normal flora of mucous membranes of the upper respiratory, gastrointestinal, and female
genital tracts.In these site it may predominate and cause superinfection.
Pathogenesis and clinical findings:
Predisposing factors to Candida infections are diabetes mellitus, general deplitity,
immunodeficiency, indwelling urinary cathters, intravenous drug abuse, prolonged treatment
with broad-spectrum antibiotics (which alter normal flora) and corticosteroids.
Clinical affection includes:
1-In the mouth, overgrowth of C.albicans produces white patches i.e. oral thrush or
moniliasis.
2-Valvovaginitis with itching and discharge which is favoured by high pH, diabetes, or
prolonged use of antibiotics.
3-Skin invasion occurs in warm, moist areas, which become red and weeping such as the
axilla, intergluteal folds, or inframammary folds most common in obese and diabetics.
4-Nails become involved when repeatedly immersed in water; as in persons involved in dish
washing. Painful redness and swelling of nail folds, thickening and loss of nails i.e.
paronychia.
5-Candida may disseminate to many organs or cause chronic mucocutaneous candidiasis
specially in debilitated children, diabetics, immunosuppressed patients or drug addicts.
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Laboratory diagnosis:
1-Direct microscopic examination: large oval gram positive budding yeast cells with
pseudohyphae.
2-Cultures are done on blood agar and Sabourd,s dextrose agar with actidione
(cycloheximide) to inhibit saprophytic fungi and chloramphenicol to inhibit bacteria. Large
cream coloured colonies develop after 2-3 days incubation at room temperature or 37ْc
.Colonies are identified by:
a) Gram stain film
b) Germ tube test: is done for confirmation the test is done by inoculating a light inoculum
from the colony in 0.5 ml human or animal serum which is incubated at 37ْc for 1-2 hr. On
microscopic examination, pseudo- germ tubes will be seen as extensions from the yeast cells
(drum stick appearance) if the organism is C.albicans.
c) Biochemical reactions may be used for species differentiation. Candida albicans ferment
glucose with production of acid and gas. API system is available.
Treatment:
Anti- fungal drugs.
Cryptococcus neoformans
C.neoformans is yeast cells with a gelatinous capsule. It is found in the excreta of birds
specially pigeons feaces. It is an opportunistic pathogen affecting mainly immunosuppressed
patients. Infection occurs by inhalation where it causes sub clinical lung infection. It may
spread systemically to the meninges causing meningitis.
Laboratory diagnosis:
1-Direct microscopic examination of CSF after staining with India ink reveals large gelatinous
capsule around budding yeast cells.
2-Cultures done on Sabourd‫׳‬s without acitidione, at 20-37 ْc show mucoid colonies which are
identified by India ink staining, biochemical reactions (urease positive).
3-Capsular antigens are detected in CSF by using anticapsular antibodies by a latex
agglutination test.
4-Antibodies are detected in patients, sera.
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Dermatophytes
The dermatophytes include 3 genera Epidermophyton ,Microsporum and Triciphyton. These
organisms affect the keratinized tissues; skin, hair and nails. They spread peripherally from
foci to produce ring – like lesions. Hence the name ringworm or tinea.
Diagnosis of ringworm:
1-Skin scales, nail and hair clippings are examined microscopically after digestion using
10% KOH Branching hyphae are detected among epithelial cells in skin or nails. In hairs,
hyphae or spores are detected. The latter may be outside and inside the hair (ectothrix) or
inside the hair (endothrix).
2- Cultures are done on Sabourad‫׳‬s dextrose agar containing actidion and
chloramphenicol and incubated at room temperature for up to 4 weeks. Colonies are
identified by:
-Morphology and colour on surface.
-Microscopic examination using lacto phenol cotton blue.
Tinea versicolor
It is a skin infection characterized by superficial brownish scaly areas on light- skinned
persons and lighter (depigmented) areas on dark –skinned persons. It has a worldwide
distribution .It is caused by Malassezia furfur.
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Antiglobulin agglutination test =Coomb's test
This test is used to determine the presence of Rh incompatibility which cause
(erythroblastosis fetalis).
About 80% of individuals have Rh antigen on their red cells i.e. they are Rh positive. Those
who do not have Rh antigen on their cells are Rh negative; and to this group the Rh antigen is
considered foreign to which they will respond by antibody formation.
The clinical importance of Rh antigen lies in the ability of the red cells of an Rh positive fetus
(inherited from Rh positive father) to induce anti-Rh antibodies in his Rh negative mother.
Fetal RBC,S enter the maternal circulation in small numbers during separation of the
placenta .The Rh negative mother will respond to these Rh positive cells by forming anti-Rh
antibodies. These antibodies will pass through the placenta to the Circulation of the fetus
during the next pregnancy, and will cause haemolysis of his RBC‫׳‬s.
This; condition is known as haemolytic disease of the new born, or (erythroblastosis foetalis).
The first child usually escapes this condition.
The condition can be prevented by injecting the Rh negative mother with a small dose of a
potent anti-Rh serum within 3 days after delivery of an Rh -positive infant. Most probably,
the injected antibody combines with the Rh positive cells in in the mother's circulation, and
diverts them from the antibody forming tissues.
Most anti-Rh antibodies are IgG incomplete antibodies which can only coat the Rh positive
RBC‫׳‬s, but cannot bridge between two RBC‫׳‬S to cause agglutination.
These antibodies can be detected by the antiglobulin Comb's test which is performed in two
ways:
1) Indirect Comb's test:
The mother's serum - containing incomplete anti – Rh antibodies -is mixed with Rh positive RBC‫׳‬S (GROUP O). After incubation at 37ْ C for 30 min. 1 h the mixture is
centrifuged, the deposit containing red cells coated with incomplete antibodies is
washed, and antihuman globulin (prepared in rabbits) is added, and the tubes
incubated. The antihuman globulin causes agglutination by linking together the
incomplete antibody molecules.
2) Direct Comb's test:
This test can detect incomplete Rh antibodies coating the RBC‫׳‬s of the new born in
erythroblastosis foetalis.
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The antihuman globulin is added directly to a washed suspension of the new born RBC‫׳‬s
Agglutination occurs.
Both the direct and indirect Comb's test are also used to detect incomplete antibodies in
autoimmune haemolytic anemias.
Virus haemagglutination
Some viruses e.g. influenza, Para influenza, mumps, adeno and yellow fever viruses can
cause agglutination of red blood cells of man ,chicken, guinea pig, rat and other animals.
This reaction is used for the detection and titration of haemagglutination viruses in culture
materials.
Enzvme - Linked Immuno Sorbant Assay (ELISA)
This technique is very sensitive and does not require' specialized equipment and avoids the
hazards of radioactivity. The method depends on conjugation of an enzyme to either antigen
or antibody, and then the enzyme activity on a substrate is used as a quantitative measure.
Solid - phase ELISA is widely used to measure antigen or antibody.
To measure antibody the indirect method is used .A known antigen is fixed to a solid phase
(e.g. plastic cup or microplate) incubated with the test serum dilution,then washed to remove
excess unattached antibody and reincubated with antiglobulin labeled with a suitable enzyme
(e.g.horseradish peroxidase).the labeled antiglobulin will attach to the antibody bound to the
fixed antigen. After washing the enzyme activity is measured by adding a specific substrate
and measuring the degree of colour change.
To measure an antigen, the double antibody technique (Sandwich technique or Direct
method) is used; a known antibody is fixed to the solid phase. The test material containing
antigen is added and the excess washed.a specific known antibody labeled with enzyme is
added .after washing a substrate is added and the enzyme activity measured colourimetrically
and related to antigen concentration.
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