sugar fermentation

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Pharmaceutical Microbiology (PHT-226)

(Practical )By

Elmutasim O. Ibnouf

Lecturer of Microbiology

Department of Pharmaceutics & Microbiology

College of Pharmacy

Salman bin Abdulaziz University


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LABORATORY RULES

&

USING OF MICROSCOPE


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LABORATORY RULES

Many of the microorganisms used in this course may bepathogenic for humans.

Certain rules are necessary to insure safety for you.1. The laboratory benches must be kept free of articles not

actually used.

2. Wear a coat during the laboratory period to protect yourclothes.

3. Smoking, eating, drinking and gum chewing are notpermitted.

4. A jar of disinfectant is provided on each bench for holding the contaminated pipettes.

5. Please return all reagents, cultures and glassware intheir respective places.


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6. A void unnecessary conversation, noise, or movement.7. Laboratory notes should include results obtained from

the experiments, a drawing of each microscopicobservation, and explanation of results reported.

8. Close doors, and windows to prevent contamination.

9. Record the results of your experiments directly in your

laboratory notebook.Do not record your results on a piece of paper with the idea

that they will

be transcribed later.

10. When you have finished using the microscope, clean

excess oil from the oil immersion lens with lens paper andxylene.

11. At the end of laboratory period, turn off the Bunsen

burner, and re-arrange your staining rack before leaving

12. Each student should have a white coat and marker.


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THE MICROSCOPE

Microbiology is the science that deals with the living organismsvery small to be seen with the naked eye, thus the advent ofmicrobiology dates from the invention of the microscope.

Types of Microscopy:1. Light Microscopy:

a. Bright field microscopyb. Dark field microscopy

The source of light set at the side of the specimen, and so thelight reaching the objective lens is the light which isreflected from the specimen. The organism appears bright

in the dark background.c. Florescent microscopy

The bacteria stained with florescent dye and U.V. light

is used.


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d. Phase contrast microscopy

Here the object appears dark in bright background.

This can be used to illustrate the structure of the

specimen.

2. Electron Microscopy:

Has a very high resolution and very high

magnification power.

a. Transmission electron microscopy (TEM)b. Scanning electron microscopy (SEM)


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Light Microscope:The compound light microscopy is composed of:Two lens system of magnification :

a. Ocular lens:Usually has a magnification of X10.

b. Objective lens:The objective lenses are of two types:

1. Dry lenses:

a. Low power objective lens, magnification is X10.b. High power objective lens, magnification is X40.2. Oil immersion lens:

Magnification is X100.Magnification:The total magnification power = magnification of the ocular

lens X magnification of the objective lens.Total magnification with low power = 10X10 = X100Total magnification with high power = 10X40 = X400Total magnification with oil immersion = 10X100 = X1000


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2. Stage:

On which the slide will be placed.

In the center of the stage, there is a hole through which theobject can be illuminated from below.

Its movement is controlled by the coarse adjustment knob

and fine adjustment.

3. Illuminating system:Its composed of:

a. Lamp.

b. Condenser

Lenses gather the light from lamp.

c. Iris of diaphragm

Regulate the amount of light passed through an opening in

the stage to the slide.


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Importance of oil utilization:Because the refractive index of air is less than that of

glass, light rays are refracted as they pass from themicroscope slide into the air. Thus many of the light rays

passed from the specimen are refracted as so great angle thatthey completely miss the objective. Placing oil that has thesame refractive index as glass between the slide and theobjective lens will greatly decrease refraction so increase theamount of light passed from the specimen to the objective.Direction for using the microscope with oil immersionlens:Procedure:

1. Place the slide provided on the stage, specimen side up,and center the section to be examined as accurately aspossible over the hole in the center of the stage.


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2. With the low power objective in position, adjust the lampuntil it gives maximum amount of light through the specimenwith the aid condenser and iris of diaphragm.

3. Rotate the nosepiece until the oil immersion objective clicksinto position.4. Place a drop of cider wood oil on the portion of the slidedirectly under the objective.5. Watching the objective from the side carefully lower it intothe oil. Dont allow the objective to touch the slide, look

through the ocular, and slowly focus with the coarseadjustment. Then, fine adjustment until a good sharp imagewill appear.6. Record your results as shown below:

Name of stain: Name of organism:

Description:Shape of cell: Arrangement of cells:Colour reaction:7. Each time after using the oil immersion lens, clean the oilfrom the objective lens with lens paper moistened with xylene.


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SOURCES OF CONTAMINATION

The purpose of this is to identify some of the sources of

contamination present in the laboratory in order to avoidthem.

Material:You have been provided with 4 Nutrient Agar plates.a. Contamination from hands:

1. Take a Nutrient Agar plate and divide it into 4 sections:a. Unwashed.b.Washed.c. Disinfected.d. Control.

2. Imprint your finger print on (a) unwashed area.3. Wash your hands throughly, dry your hands in air and

imprint the same finger prints on (b) washed area.


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4. Wash your hands with soap, water and disinfectant, dryyour hands in air, do not use hand towel. Imprint the samefinger on the (c) disinfectant area, leave section (d) ascontrol.5. Incubate your plates for 1 day at 370C and record theappearance of the plate.b. Contamination from breath:1. Take a Nutrient Agar plate.

2. Hold it in front of your mouth.3. Cough and breath vigorously.4. Invert the plate and incubate for 1 day at 370C.5. Record the appearance of the plate.c. Contamination from air:1. Expose a Nutrient Agar plate to the air on the bench forone hour.2. Invert the plate and incubate for 1 day at 370C.3. Record the colonial appearance.


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EXAMINATION OF THE MICROORGANISMS

UNDER THE MICROSCOPE

There are two ways in which the microorganisms may beexamined under a microscope, in the living state or inthe fixed state.

1. In the living state:

a. Wet Mount:The purpose of this exercise is the examination ofliving microorganisms in a liquid phase

I. It is used to observe certain activity e.g. : Reproduction MotilityII. Used in case where staining and other manipulations

affect the structure of microorganisms.


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Materials: Suspension of microorganisms in a mixed culture

bottle, marked (W). Slides and cover slips.

Method:1. Take a clean slide, pass through the flame of the

bunsen burner.2. Add a loopful of culture on to the centre of the slide.

3. Carefully place a cover slip over the drop.4. Look under the microscope under the dry lenses (X10and X40).

5. Record the results.b. Hanging Drop Preparation:

The object of this exercise is to observe themicroorganism and test of its motility.


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Materials: Culture (M) and (N) (One is motile and the other is

non-motile). plastacine.

Slide and cover slip.Method:1. Take a clean slide.2. Roll plasticine in between your palms to make a thin

elongated roll, and make a ring out of it.3. Place it over the slide making a circle, smaller in size than

the cover slip.4. Place drop of bacterial culture in the centre of cover slip,

by a loop.5. Invert the slide over the cover slip and gently press the

slide against the plasticine ring.

6. Turnover the slide quickly, so that the cover slip is on topand the slide at the bottom, allowing the drop to hang.7. Look under the dry lens X10 and X40 later, record your

observations.


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2. In the fixed state:Staining:

Most microorganisms are transparent, so special

techniques are used to visualize microorganisms, one ofthose techniques is staining.

1. Staining enhances the morphological appearance.2. It is used to identify structural parts.3. It is used to differentiate microorganisms (e.g. Gram +ve

, Gram -ve).Smear preparation:The purpose of this technique is to prepare the

microorganism for staining and preventing them frombeing washed during the process of staining.

a. From liquid culture:

1. Take a clean slide, and pass through the flame2. Place a loopful of culture in the centre of the slide and

spread evenly.


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3. Allow it to dry in air.4. Pass through the flame three times quickly to heat kill

and fix the organism to the slide.5. Smear is ready to be stained.b. From solid culture:1. Take a clean slide and path through the flame.2. Place a loopful of saline in the centre of the slide.3. Take a little amount of solid culture on to the loop,

emulsify in the saline evenly.4. Allow it to dry in air, and pass through the flame toheat kill and fix the organism to the slide.

5. Smear is ready to be stained.6. Stain the slide and record the results.


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TYPES OF STAINING

Simple Staining:The object of this exercise is to see the shape,size of different microorganisms, protozoa and fungi,

by using a simple stain like Methylene Blue, a dye tostain microorganisms.

Materials:

A basic dye (methylene blue). Bacteria (Staphylococci) labled (S). Fungus (Candida) labled (C).Method:1. Make a smear of the microorganism provided, as

mentioned previously.2. Place the smear on to the staining rack.3. Add methylene blue stain (for 2 minutes).4. Wash the strain off the slide with water.5. Blot the smear with filter paper.


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6. Dry the slide, put the oil and look under the oilimmersion lens (X100).

7. Record your observations.

Negative staining:Certain microorganisms are very difficult to

stain (e.g. Spirochetes), and can be visualized bystaining the background (negative staining) by certain

dye (India ink, nigrosine) leaving the cell transparent,negative staining will not give any information aboutthe cell contents, however the cell shape and size areeasily determined.

Materials: Culture of Bacillus species (marked B).Nigrosine stain.


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Method:

1. Take a clean slide and place a drop of Nigrosin at

one end of the slide.2. Emulsify organisms in Nigrosin.

3. Using the edge of another clean slide spread the

drop out into a film ( like a blood film).

4. Allow it to dry in air and examine under oilimmersion objective.


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Differential Staining:

It is used

More thato differentiate between microorganisms or parts of

same cell. n one dye is used in differential staining.1. Gram staining:

Discovered by Christian Gram in 1884. Thisstaining process divides bacteria into two groups, Gram-

positive bacteria which retain the blue/violet color afterdecolourization by alcohol and colored blue/violet.Gram-negative bacteria loose their blue/violet color aftertreatment with alcohol and color red by counter staining.

Material:

Bacteria labeled as (P) and (N). Gram stain.


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Method:1. Prepare the smear as mentioned before.2. Place the slide on the slide rack.3. Add crystal violet for 1 minute, then wash with water.4. Add iodine and leave on the slide for 1 minute, then

wash with water.5. Decolorize with alcohol for 10 to 20 seconds, then wash

with water.

6. Add safranin on the slide for 30 seconds, then wash withwater, blot dry.

7. Examine under the oil immersion lens (X100).

8. Record your results.


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2. Acid-Fast Staining (Ziehl-Neelsens Stain):

Acid-fast bacteria (Mycobacteria) aredifficult to stain by ordinary dye (due to high lipid

contents), so special techniques should be applied in

order to stain such bacteria. For example applying of

heat will facilitate the penetration of specific dye(Carbol-fuchsin). Once the cells are stained they

retain the dye even after a very strong decolorizing

agent (acid alcohol) have been used. This treatment

decolorize other bacteria.

Materials:

Culture of Mycobacteriumplate labeled as (Mb).

Ziehl-Neelsens stain.

Alcohol, saline.


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Method:1. Prepare smear of Mycobacterium.

2. Allow the film to dry in air. Heat fix the smear.3. Cover the slide with concentrated carbol fuchsin.4. Gently heat, till the steam rises.5. Wash the slide with tap water.6. Decolorize the smear with acid alcohol for 10 to 30

seconds.7. Wash the slide with tap water.8. Apply methylene blue for 30 to 45 seconds. Wash and

blot dry and examine under oil immersion objective.


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Spore Staining:Introduction:

Certain bacteria (e.g. Bacillus, Clostridium) formendospores. Bacterial spores do not take up stains readily andconventional techniques merely stain the vegetative portionsof the cell, leaving the spore as a clear one. By vigoroustreatment (e.g. strong stains and prolonged heat) it is possibleto stain the spore but, once introduce, the stain is resistant to

decolorizing agents. The latter bleach the vegetative parts ofthe cell, which may then be counter-stained. The presence ofspores, their size, shape, position and whether they bulge thewalls of the parent cell are important characters in theidentification of bacteria.Materials: Culture of Bacillus species marked (B). Malachite green. Safranin.


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Method:

1. Prepare and fix the smear with bacteria provided.

2. Cover the slide with malachite green stain.3. Gently heat until you see steam rising (DO NOT BOIL

THE SMEAR).

4. Allow the stain to remain for 5 minutes.

5. Wash with tap water.

6. Counter-stain with safranin for 30 seconds.

7. Blot dry, and look under oil immersion objective.


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Microbiological Culture Media

Cultivation of microorganisms in the laboratory is essentialfor the study of their morphological and physiological

properties, also for their isolation in pure form andidentification. This cultivation is possible by using anartificial medium.

The medium may be in liquid (broth) form, solid (containing

agar) form or semisolid (containing low concentrationof agar as solidifying agent) form.Media may be classified on the basis of content into

complex, chemically defined (synthetic) and livingmedia.

1. Complex Media:Contain extract or digest of animals (beef or meat extract),

plants (soya bean digest), or microbes (yeast extract)that supply all the essential nutrients.


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Chemically Defined (Synthetic) Media:Essential nutrient is provided by a pure chemical of known

composition e.g. K2HPO4 , KH2HPO4 , (NH4)2SO4 ,

glucose, etc3. Living Medium:

Designed specifically for obligate intracellular

microorganisms e.g. viruses.

Complex media may be classified as follow:1. Ordinary or Basic Media support the growth of

ordinary microorganisms not a fastidious one:

a. Nutrient Broth is the basis for most media used for

growth of microorganisms, it contains: meat extract,

peptone and sodium chloride.


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b. Nutrient Agar is nutrient broth solidified by

1.5-2% agar. It is the basis for other solid selectiveor enriched media.


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2. Enriched Media certain organisms e.g. gonococcus,

pneumococcus are so nutritionally that they cant grow

on the ordinary media. They require more complexorganic body fluids for their growth. Media containing

such substances are called enriched media, for example:a. Blood Agar:

-It is a nutrient agar enriched with sterile blood (5-

10%).-Blood agar is very useful not only as an enrichedmedium supporting the growth of most fastidiousorganisms, but also as indicator medium, differentiatingorganisms according to their action on the blood.

-Some causes complete haemolysis and those arecalled -haemolytic bacteria.


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-Other causing greenish coloration or partial haemolysis

and those are called - haemolytic bacteria.-Others producing no change, e.g. gamma-haemolyticbacteria.


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b. Chocolate Agar: (or heated blood agar):- Prepared from blood agar by heating of the melted

blood agar at 1000

C for 2 minutes.-Used particularly for the culture of the haemophilusgroup of organisms.


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3. Enrichment Media:

-They are fluid media-They contain some substances which support thegrowth of most microorganisms e.g. Thioglycolatemedium, or substances which inhibit the growth ofunwanted organisms. For example:

* Fluid Thioglycolate Medium:-This medium supports the growth of both aerobic,microaerophilic and anaerobic bacteria.-It contains sod.Thioglycolate, cysteine and dextroseas reducing agent.

-Aerobic bacteria will grow near the top whereasanaerobic grow at the bottom and microaerophilicgrows in between.


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* Selenite Broth: used for isolation of shigella andsalmonella groups from faeces.

Tetrathionate Broth: used for isolation of salmonellacausing food poisoning.


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4. Selective Media:

a. Mannitol Salt Agar (MSA):

- This medium is a selective medium for pathogenicstaphylococci.

- It contains salt (7.5%) which will inhibit the growth

of other microorganisms except staphylococci.

- Also contain mannitol as a test sugar and phenol red

as indicator.

- Staphylococcus aureusferments mannitol, producing

yellow colonies.


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b. Sabouraud Dextrose Agar (SDA):

- It is a selective media for fungi, it contains high

concentration of dextrose, chloramphenicol to inhibit thegrowth of bacteria and has pH of 5.56.

5. Selective and Differential Media:

-Selective media contain some dye or other chemical that

inhibits the growth of certain organisms, and allow the

growth of others.

-Differential media usually contain an indicator which

changes its color with certain organisms and not with others.

MacConkeys Medium:

- It is used in the detection and isolation of all types ofenterobacteriaceae from stools.

-It contains bile salts which will inhibit the growth of all other

microorganisms except that of enterobacteriaceae.


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Also contains lactose as a test sugar and neutral red as

indicator. On this medium the lactose fermenters produce a

rose pink colony, and the non-lactose fermenters produce apale yellow colony.


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6. Media for Anaerobic Bacteria: Cooked meat medium.

Fluid thioglycolate medium.


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Materials:A. Liquid culture of :

1. E.coli2. B.subtilis3. C.albicans4. C.perfringens5. S.aureus

B. Media:1. Nutrient broth2. Nutrient agar

3. Thioglycolate broth

4. Blood agar

5. Sabouraud agar6. Mannitol salt agar


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Procedure:

1. Examine each medium for content and colour.

2. Inoculate the media with the exact microorganism

following the table.

3. Incubate for 24 hours at 370C.

4. Record your results in the table.

5. Make a wet mount preparation from the cell culture and

examine it under microscope, draw the shapes of thesecells.


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CULTIV TION OF MICROORG NISMS

Inoculation with the loop:

The object to illustrate how to handle the loop ininoculation and the effectiveness of loop flaming.

Materials:- 3 tubes containing 5 ml of media.- Culture of Bacillus subtilis lablled B.

Procedure:1. Lable the tubes 1, 2, 3 .2. Pick up the culture (B) in your left hand between your

fore and middle fingers.3. Pick up the loop in your right hand.

4. Sterilize the loop by flaming until it becomes redhot.5. Remove the cap of the culture by the little finger of your

right hand.6. Introduce the loop into the tube containing the culture

(B) withdraw.


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7. Flame the mouth of the tube and cap.

8. Transfer a loopful of the culture to tube (1).

9. Take another loopful from the culture, flame theloop,and inoculate into tube (2).

10. Leave tube 3 as control.

11. Incubate all tubes at 370C for 1 day.

12. Report your results.


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Isolation of bacteria by streaking technique:

This method may be used for checking the purity of a bacterialculture, and may also be used for isolating individualspecies from a mixture. Culture with single type of colonyis regarded as a pure culture.

Materials:Nutrient Agar plates.

Bacterial culture marked (B).Procedure:1. With a sterile loop, transfer a loopful on to the surface of a

nutrient agar plate. Place the drop near the edge.2. With the trailing edge draw the loop tightly over surface in

parallel line.3. Flame the loop.4. Turn the plate at a 900 angle and repeat the streaking across.


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5. Flame the loop.

6. Repeat (4) & (5) again at right angle.

7. Label at the back of the plate.8. Invert the plate and incubate for 1 day at 370C .

9. Examine and describe the appearance of the growth of

the colonies on the plate.


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acterial Count

Measurement of the bacterial growth in liquid medium can be

done by:

I. Measuring the cell number.

II. Measuring the cell mass.

III. Measuring the bacterial metabolic activity.

(I) Measuring the cell number: (Direct count)This can be done by the following methods:

1. Measuring the total cell number by:

a. Using the counting chamber:

The counting chamber is divided by lines into

small squares by counting the number of organisms in all

squares, then the number of organisms per milliliter can

be calculated by multiplying the counted number by

dilution factor. This is the total count of live and dead

organisms.


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b. Using electronic cell counter (Coulter counter):

The electrical resistance of the fluid in a small

hall is measured, as each cell passes through the hole,

the resistance increases highly and the increase isrecorded. Also this is the total count of both live and

dead cells, but t is faster method than the previous

method.

2. Measuring only the live cell (Viable count):

This method is used to determine the number of

living bacterial cells. The principle of this method is

based on the ability of each cell to give rise to a colony

if it is allowed to grow over solid medium.

Three methods are used in this determination of viablebacteria:

A. Spread plate method.

B. Pour plate method.

C. Dropping on Agar surface.


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(A) Spread plate method:In this method a given volume of sample is

serially diluted and the small amount from highest

dilution is pipetted on to surface of hardened NutrientAgar. Then spread with glass spreader.(B) Pour plate method: From the sample diluted as mentioned above, a

constant amount is added from each dilution into a

sterile plate and molten Agar is added and mixed well inthe plate and Agar allowed to set.(C) Dropping on Agar surface:

Miles and Misras technique:Here also the sample should be serially diluted,

with a standard 50pipette add 5 drops from eachdilution on the well dried nutrient agar plate. Allow thedrops to dry. After each experiment incubate the plates at370C for 24 hours. Count the number of colonies andmultiply by the dilution to give you the organism / ml in

original culture.


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Determination of viable count:Materials:1. Bacterial culture (E.coli).2. 7 bottles each containing 9 ml Ringer solution.3. 1ml pipettes (7).4. Petri dish plates (3).5. 50 l dropper.6. Nutrient Agar plates (3).

7. 319 ml molten nutrient agar.Method:1. Label the bottles from 1-7 .2. Make a serial dilution as follows:

a. Discard the used pipette, then by using a sterile

pipette mix the contents of bottle # 1 and remove 1 mlinto bottle # 2 .

b. Repeat this step up to bottle # 7 .


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3. From the last 3 dilutions (10-5, 10-6, 10-7( do the

following:

A. Pour plate:I. Label 3 petri dishes matching the 10-5, 10-6, 10-7

dilution.

II. From each dilution transfer 1 ml of the diluted culture

to petri dish then add 19 ml of melted Agar and mix

well.

III. Wait until the Agar solidify, then incubate at 370C for

24 hours. Count the No. of colonies / dilution.

IV. Calculate the # of CFU / ml.


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B. Surface viable count:

I. Label 3 nutrient agar plates of the dilution (10-5, 10-

6

, 10-7

(.II. Using the dropper (50) l starting from (10-7(

dilution transfer 5 drops to each Plate, then go to the

next dilution (10-6( using the same dropper. Repeat

that with final dilution (10-5( .

III. Wait till the drops dry. Incubate at 370C for 24 hours.

IV. Count the colonies in each drop for average

concentration. Take the average of 5 drops.

V. Calculate the # of CFU / ml.


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(II) Measuring the cell mass:

A. Turbidimetric method:

This method depends on the change in theoptical density that occurs due to the increase in the

number of bacterial cells. The number of cells is

directly proportional to the amount of light

absorbed.

B. Determination of net weight:

This is done by centrifuging the cells and

weighing the pellet of cells obtained.

C. Determination of dry weight:

It is done by drying the centrifuged cellsmass before weighing by placing it overnight in an

oven at 100-1050C.


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(III) Measuring the bacterial metabolic activity:

By measuring either the metabolic products

e.g., acids or the decrease in the substrates that areconsumed in the metabolic activity e.g. sugars.


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EFFECT OF ENVIRONMENT L CONDITIONS ON

B CTERI L GROWTH

A. Effect of temperature on bacterial growth:Temperature is one of the most important factors

influencing growth of bacteria since temperature effectenzymes activities. In this exercise the effect of different

temperatures on growth will be examined.Materials:

- Culture of Echerichia coliand Bacillus subtilis.- Eight Nutrient Broth.

Methods:

1. Label the tubes as follow:-E.coli(1-4).-B.subtilis(5-8).2. Inoculate tubes (1-4) with E.coli.3. Inoculate tubes (5-8) with B.subtilis.


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4. Incubate the tubes as follow:

5. Record your results.

Tubes No. Temp.1,5 50C

2,6 250C

3,7 370C

4,8 450C


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B. Effect of pH on bacterial growth:

pH effects bacterial growth since it limits the activity

of enzymes required for bacterial growth.Materials:

- Culture of E.coli, S.aureusand C.albicans.

- Nine Nutrient Broth at pH 3, pH 7, and pH 9 .

Method:

1. Label tubes with the proper bacterial name at differentpH.

2. Inoculate each tube with the labeled bacteria.

3. Incubate at 370C for 24 hours.

4. Report your results and comment.

C Eff t f ti b t i l th


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C. Effect of osmotic pressure on bacterial growth:

Cell walls protect prokaryotes against changes in

osmotic pressure over a wide range. However, sufficiently

hypertonic media at concentrations greater than those insidethe cell (such as 20% sucrose) cause water loss from the cell

by osmosis. Fluid leaves the bacteria causing the cell to

contract, which, in turn, causes the cell membrane to separate

from the overlying cell wall. This process of cell shrinkage is

called plasmolysis.

When bacteria are placed in hypotonic media with

concentrations weaker than the inside of the cell, water tends to

enter by osmosis. The accumulation of this water causes the

cell to swell and then to burst, a process called osmotic lysis.Materials:

Culture of E.coliand S.aureus.

Nutrient broth at 0.9% NaCl and 5% NaCl (2 each).


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Methods:

1. Label tubes with proper bacterial name at different

salt concentration2. Inoculate each tube with the labeled bacteria.

3. Incubate at 370C for 24 hours.

4. Report your results.

D Eff t f h t


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D. Effect of heat:Destruction by heat:- Non-spore forming bacteria (vegetative cells) can not

withstand temperature at 60

0

C within 30-60 min.- Spore forming bacilli can withstand this temperature andcan withstand boiling from few minutes to 3 hours butwill kill at 1200C at 2 atmosphere steam pressure within20-30 minutes.

- In this experiment you will compare the heat resistanceof Bacillusas example of spore forming bacteria andS.aureusas example of vegetative cells.

Materials:1. Nutrient broth 15 ml each (6 tubes).

2. Culture of S.aureus(S) and of Bacillus subtilis(B).3. Nutrient broth 5 ml each (30 tubes).4. 3 water baths set at 600C, 750C and 1000C.


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Procedure:

1. Divide the six large tubes into 2 groups each containing

3 tubes.2. 0.5 ml of the culture (S) is transferred to each tube of

one group, and 0.5 ml of culture (B) is transferred to

each tube of the other group.

3. A tube of each group is taken and placed in water bathset at 600C, 750C and 1000C respectively.

4. On specific time interval 0, 5, 15, 30 and 60 min., one

loopful of each tube is transferred to a labeled tube of 5

ml Nutrient broth.

5. The tubes of 5 ml Nutrient Broth are collected andincubated at 370C overnight.

6. Read the results and tabulate as follows:


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Effect of Heat on Bacteria at 600C

60 Min.30 Min.15 Min.5 Min.0 Min.Time

Organism

S.aureus

B.subtilis


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Organism

S.aureus

B.subtilis


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Organism


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CONTROL OF MICROBI L GROWTH

Microbial growth can be controlled either by:

-Complete killing of all M.O (sterilization)

-Or inhibiting the growth of M.O.

Sterilization is an absolute term. This means that the sterile

substance is completely free from any M.O.

The methods used for sterilization are classified into:-Physical methods.

-Chemical methods.

Physical methods of sterilization:

1. Heat (Dry & Moist) 2. Radiation (U.V. light) 3. Filtration

1 D H


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1. Dry Heat:

The thermal death of M.O. takes place due to

inactivation of essential cellular proteins or enzymes

through oxidation.Methods:

a. Incineration e.g. platinum loop.

b. Flaming e.g. mouth of culture tubes.

c. Hot air oven e.g. 1600

C for 1 hr.- Sterilization of dry glass wares as test tubes, flasks

pipette, petri dishes ..etc.

- Sterilization of fixed oils and powders.


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2. Moist Heat:

The thermal death takes place through coagulation.

Methods:A. At temperature below 1000C:- Pasteurization of milk (630C for 30 min.[holdermethod] or 720C for 20 sec.[flash method]).

Such temperature is enough to kill any pathogenicM.O.that can be transmitted by milk e.g.

Mycobacteria, Salmonella and Brucella.- Tyndalization 560C for 1 hr on several successivedays. This method is used only for thermolabilesubstances in which bacterial spores can germinate

between the first and second heating as milk, serum

and body fluids.B. At temperature of 1000C:1. Boiling at 1000C for killing of non-spore forming

M.O.2. Steaming at 1000C e.g. sterilization of sugar media

and gelatin media either by:

I. Steaming for 90 min.


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II. Steaming for 20-40 min on three successive days.

3. Heating with bactericide e.g. 0.2% chlorocresol or

0.002%phenyl mercuric nitrate

C. At temperature above 1000C e.g. Autoclaving:

- Autoclaves are widely used in sterilization of culture

media, surgical supplies and many other instruments.

- A complete sterilization can occur at 15 Ib per sq. in

guage pressure at 1210C for 15 min.Sterilization by Radiation:

1. Ultraviolet light:

- The ability of sun light to kill bacteria is mainly due

to the ultraviolet rays.- The germicidal activity of ultraviolet light increased

as it is wavelength () decrease and intensity increase.


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Ultraviolet radiations can be produced by mercuryvapour lamps

- The lethal effect of U.V. rays is due to it is effect onthe DNA of the cell through the formation of

pyrimidine dimer on the same strand.- Ultraviolet rays are used for sterilization of air (e.g.

operating theatres), fluids and punches.2. Ionizing radiation:- They are very lethal to cells, and their lethal effect is

due to either direct ionizing of the cellular DNA orby the chemical effect of ionized water, e.g. -rays

which have a very high penetration power, andwidely used for sterilization of plastics (e.g.disposable syringes, pipettes and dishes) foodstuffsand drugs.

S ili i b fil i


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Sterilization by filtration:- Fluids can be rendered free from bacteria by passing

through special filters.

This method is used in making sterile preparations ofthe soluble products of bacterial growth such astoxins, and for sterilization of liquids that would bedamaged by heat such as serum and antibioticsolutions also for oil damage by heat.


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CHEMICAL METHODS USED FOR

MICROBIAL CONTROL

The microbial control can be achieved by using chemical

antimicrobial agents.

Definition of some terms:

Bactericide: An agent which kills bacteria.

Bacteriostat: An agent which inhibits the microbial growth.Fungicide: An agent which kills fungi.

Fungistat: An agent which which inhibits the growth of

fungi.

Disinfectant: Is a substance which has the ability to kill M.Owhen applied to the surface of an inanimate object.


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Antiseptic: Is a substance which has the ability to kill M.O.

on living tissues.

Preservatives: Are agents which are used in food stuffs andmany pharmaceutical preparations to prevent microbial

spoilage of the product and to minimized the risk of consumer

acquiring infection when the preparation is administered.

Evaluation of the antimicrobial agents:A. Determination of MIC:

The principle of evaluation is based on determining

the minimum inhibitory concentration MIC of the agent.

MIC: Is the lowest concentration of the agent which inhibits

the growth of a given microorganism in a given time.


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Methods which can be applied to determined MIC:

1. Serial dilution in broth (Broth dilution method):

A serial dilution of the agent is made in broth,

and the tubes are inoculated with the test organism

and incubated. The lowest concentration at which no

growth occurs in taken as the minimum inhibitory

concentration (MIC).2. Serial dilution in agar (Agar dilution method):

In this method the dilutions of the substanceunder test are made in agar instead of broth. The agarcontaining the substance under test is subsequently

poured onto petri dish. This method has theadvantage over the previous one in which on a singleconcentration of tested substance, several organismsmay be tested.


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Determination of MIC by broth dilution method:Materials:

A standard solution of disinfectant / antibiotic . 9 ml Mueller-Hinton broth tubes (5). (E.coli) culture labeled as (E).

Procedure:

1. Label the 5 broth tubes from 1-5 with marker.

2. Add 1 ml of disinfectant to tube 1and mix .3. Add 1 ml from tube 1to tube 2 and mix .

4. Continue dilutions in this manner to tube 5 .

5. Add 50 l E.colibroth culture in each tube with a

dropper.6. Shake well the tubes and incubate at 370C for 24

hours.

7. Record the results.


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Susceptibility by disk diffusion method:

The susceptibility of tested microorganism to

the discs of filter paper impregnated in differentantimicrobial agents is tested, and the zone of

inhibition are observed.

Materials:

Mueller-Hinton agar plates.

Antibiotic discs.

E.coliculture labeled (E).

Procedure:

1. Using sterile cotton swab inoculate the surface of

Mueller-Hinton agar plate with bacterial culture (E).2. With sterile forceps apply the different discs of

provided antibiotics to the surface of the agar plate.


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3. Incubate at 370C for 24 hours..

4. Measure the zone of inhibition in mm of each disc

using the bottom of the plate.

5. Record the results.


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Evaluation of disinfectants (Determination of Phenol

Coefficient):

This is a method used to measure the bactericidal

power of disinfectants. Phenol is used as the standard and

the agents activity is compared to phenol ability that kills

standard culture of bacteria at specific time.

The Rideal-Walker test:

Materials:-Phenol solution 10% .

-Disinfectant solution 1% .

-Culture of E.coli.

-Nutrient broth tubes (40).-Sterile test tubes (20).


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Method:

1. Label 5 tubes and make 5 dilutions of phenol with

distilled water as follows:1/95 , 1/100 , 1/105 , 1/110 , 1/115 .

2. Take an other set of test tubes (5 tubes) and dilute

the disinfectant with distilled water to give the

following dilution:

1/100 , 1/200 , 1/300 , 1/400 , 1/450 .

3. Label 5 sterile test tubes A, B, C, D and E . From

each phenol dilution add 5 ml to each tube.

4. Take an other set of sterile test tubes (5 tubes) and

label them K, L, M, N and P . To each tube add 5ml of the different concentration of disinfectant.


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5. For each set of tubes (phenol disinfectant) the

following steps are followed:

6. Take 20 tubes of nutrient broth and arrange them in arack in four rows of five. Label them as below:

A 1- 4 K 1-4

B 1- 4 L 1-4

C 1- 4 M 1-4

D 1- 4 N 1-4

E 1- 4 P 1-4

7. Take 1 ml from the culture add 0.2 ml to tube (A) then

at 30 second interval add another 0.2 ml to tube (B).Continue the addition every 30 seconds until tube (E).

(Avoid touching the wall of the tube).


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8. After 30 seconds from the previous step, using a loop,

transfer a loopful from tube (A) to (A1).

9. Repeat this step with tube (B), then (C), (D) and (E).10. Then transfer another loop from (A) to (A2) and

repeat for the other.

11. Continue this treatment until tube (E4).

12. The same procedure is done for (K) - (P) tubes.13. Incubate all broth tubes for 42-48 hrs at 320C.

14. Examine the tubes for growth and record the result as

positive and negative in the provided table.

15. Phenol coefficient is the dilution which shows growth

in 2.5 minutes and 5minutes but not in 7.5 minutes

and 10 minutes, divided by the phenol concentration

which shows the same results.


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A B C D E

2.5 A1 B1 C1 D1 E1

5 A2 B2 C2 D2 E2

7.5 A3 B3 C3 D3 E3

10 A4 B4 C4 D4 E4


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Exposure time (minutes)

2.5 5 7.5 10

DilutionBacteria

- - - -

+ - - -

+ + - -

+ + + -+ + + +

1/95

1/100

1/105

1/1101/115

Phenol

- - - -

+ - - -

+ + - -

+ + + -

+ + + +

1/100

1/200

1/300

1/400

1/450

Tested Disinfectant


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R-W Coefficient:tD dilution of disinfectant kill M.O. in 7.5 min. not at 5 min. 300

__ = _________________________________________ = ____ = 2.9

pD dilution of phenol which kill M.O. in 7.5 min. not at 5 min. 105

St ilit T ti


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Sterility Testing

Sterile object is an object that is completely free from all

living organisms e.g. bacteria, fungi, virus, etc. The term

sterile is an absolute term, i.e., the material is sterile or not.

Sterility test is used to insure the sterility of the object like

liquids that will be injected into the body, ophthalmic

preparations, sutures, and surgical dressings. Sterility tests are carried out on statistically significant

random samples of the batch and carried out for bacteria

and fungi only.


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Media for bacteria:For both aerobic, anaerobic and microaerophilic bacteria

fluid thioglycolate broth is used. Incubated at 35 37 C

up to 10 days, checked daily.

Media for Fungi:

Sabouraud dextrose broth (SDB) is used. Incubate at 22

25 C, checked daily.

Positive control:

o Fluid thioglycolate media are inoculated with

staphylococcus aureus as aerobic bacteria andClostridium histolyticum as anaerobic bacteria.

o Sabouraud dextrose broth is inoculated with Candida

albicans.


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Negative control:

o Both of the media used above with out inoculationare incubated as negative control.

Materials:

Ampoule of 2 ml distilled sterile water.

Sterile Sabouraud dextrose broth.Sterile fluid thioglycolate.

Procedure:

I. From the ampoule of the sterile distilled water 1 ml

is transferred to the Sabouraud dextrose broth and the

other 1 ml is added to the fluid thioglycolate medium.ii. Positive and negative controls are also made.

iii. Inoculate and check the result.

sugar fermentation

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