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Laboratory Manual of Pharmaceutical microbiology

BACHELOR OF PHARMACEUTICAL TECHNOLOGY

Second Year First Semester

Name: …………………………………………….

Roll No: …………………………………………

Regn. No:…………………………………………

Session: ………………………………………………

Department of Pharmaceutical Technology

JIS UNIVERSITY

81, Nilgunj Road, Agarpara, Kolkata - 700109

Department of Pharmaceutical Technology, JIS University


INDEX

Expt No Name of experiment


1 Subculturing bacteria and fungus
2 Nutrients slabs and slant preparation
3 Simple staining
4 Gram Staining
5 Acid fast staining
6 Isolation of pure culture of microorganisms by multiple streak
plate technique
7 Motility determination by hanging drop method
8 Bacteriological analysis of water
9 Imvic test

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INDEX

Date Serial Expt Name of Experiment Page Signature Marks


No No No (Out of 10)
1 Subculturing bacteria and fungus
2 Nutrient slant and slab preparation
3 Simple staining
4 Gram staining

5 Acid fast staining

6 Isolation of pure culture of


microorganisms by multiple streak plate
technique
7 Motility determination by hanging drop
method

8 Bacteriological analysis of water

9 Imvic test

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EXPERIMENT NO. - 1

SERIAL No: DATE:

SUBCULURING OF BACTERIA AND FUNGUS

AIM / OBJECTIVE

To study the technique of subculturing of bacteria and fungus.

PRINCIPLE:
In most of the labs, microbes are generally grown in solid agar media or liquid media. It is important to
revive new cultures of microbes former to using them in the laboratory exercises. These growth of
microbes are to be maintained for it’s longer use. This can be done through the subculturing the
organisms.
Aseptic technique of transfer of microorganisms from a used media to a freshly prepared medium. This
freshly medium is incubated at organism growth temperature for it’s revival.
On solid media: the transmission of bacteria or fungi from on agar slant or petri plate containing growth
medium.
From solid to liquid media: transmission of bacteria or fungi from on agar slant or petri plate containing
liquid growth medium.
From liquid to soild media: transmission of bacteria or fungi from on liquid growth medium on petri
plate containing growth medium.
From Liquid to liquid medium: the transfer of bacteria or fungi from a liquid growth culture to other
liquid growth culture.

MATERIALS REQUIRED:
GLASSWARES:
Test tube
Petri plates
Glass rod
Inoculation needle for stab culture technique

INSTRUMENTS REQUIRED:
Laminar air flow
Autoclave
Incubator
Burners
Wire loops

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Lighter/Match sticks

CHEMICALS REQUIRED:

Growth media for microbes


70% ethanol
Distilled water

PROCEDURE:
Generally the subculuring of bacteria is done by different methods depending type of organism i.e. either
it is aerobic or anaerobic microbes.
Method for sub- culturing aerobic organism
1. Spread plates
2. Streak plate

STREAK PLATE METHOD:


It is fast and quick isolation method for microbe subculture. It is one of the important technique in which the
loopful of microbial culture will be grown on nutrient media comprising agar in it. These will lead to the
spreading of the whole viable cells and individual cells will be grown as lone colony/single cell on the media.

SUBCULTUTING BY STREAKING:
1) Prepare the fresh growth media for the microbes in case of mesophilic bacteria we can use the nutrient
agar/broth, for common fungus and yeast potato dextrose agar can be used.
2) Sterilise the media using autoclave (wet sterilisation technique)
3) After sterilisation fill the test tubes/petri plates with the growth media for the microbes.
4) Let the media to be cooled at room temperature.
5) Red hot the wire loop for inoculation
6) Take of loopful of culture and streak it on the media filled in petri plates or in test tubes.
7) In case of test tubes for preparing slants, tilt the test tubes at 45 degrees for usage.
8) Incubate the petri plates/slants at growth temperature of the microbes in incubator.

DIFFERENT METHODS FOR STREAKING:


Sector streaking
Four flame streaking

SPREAD PLATE TECHNIQUE:


Principle of the spread plate technique is same that of the streak plate technique but in this techniques
isolation of micorbes is not done directly by applying culture on to the growth media but instead it is first
diluted externally either in distilled water or buffer and then culture is applied on growth medium for getting
isolated colonies.

CRITICAL QUESTIONS:

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REFERENCE:
Attendance Observation Performance Record Pretest Critical Viva Total
10 4 4 Maintenance 4 Thinking 10 40
4 4

SIGNATURE OF THE TEACHER

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EXPERIMENT NO. - 2

SERIAL No: DATE:

PREPARATION OF NUTRIENT SLAB AND SLANT

AIM / OBJECTIVE

To prepare nutrient slab and slant..

PRINCIPLE:

Agar is jellylike substance derived from purifying the cell walls of red algae. It is added to
microbiological media for solidification purposes. It has no nutritional value, so when it is used in
microbiology to culture microorganisms, various nutrients are added to increase bacteria growth in Petri
dishes or test tubes. When a test tube is used for storing the bacteria, it is referred to as an agar slant
since the liquid culture solidifies while the tube is in a tilted position. A screw-cap top on the slants
prevents the agar from drying out.

PROCEDURE:
MEDIUM PREPARATION:

The medium is prepared differently for slants than Petri dishes. Sterilization is done with the agar in the
tubes; Petri dishes are pre-sterilized before sterilized agar is poured into them. Measure the amount of
water needed and put it in a pot. Heat it on a stove until it is almost boiling. Add dry ingredients and stir
the mixture slowly until they dissolve. Before adding agar, mix it with a small amount of cold water to
prevent lumping. Use caution when adding agar to the hot liquid since it can foam and overflow the pot.
Add small amounts of agar at a time and stir to evenly distribute the agar. Turn off the heat after
bringing the agar to boil.
STERILIZING TUBES:

Place test tubes without the caps on a test tube rack. Fill the test tubes by transferring about 5 milliliters -
- about .17 ounce or 1 teaspoon -- of the molten agar from the pot using a pipette, a small funnel or a

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syringe. Place all the caps loosely on the test tubes -- the agar won't be sterilized if they are sealed tight -
- and sterilize all the tubes for about 25 minutes at 250 degrees Fahrenheit.

SLANTING:

When the agar is still hot, tilt the rack holding the test tubes on a solid surface or a thick book, making
sure the medium inside the tubes is at a slanted position. Allow the medium to cool and solidify at this
angle, which increases the surface area of the agar.
STORAGE:

Tighten the caps of the test tubes after the agar has cooled. The slants are ready for use once the agar has
solidified. They can be stored at room temperature or in the refrigerator for future use.
INOCULATION:

Inoculate the slant by transferring cells with an inoculating loop from a single-colony microorganism on
a plate to the slant's surface. Move the loop across the surface of the slant and cap the tubes. Incubate the
slant until there is evidence of growth, then put the tube in a refrigerator.

OBSERVATION:

Nutrient slab and slants are prepared.

RESULT: Nutrient slab and slants are prepared and inoculated.

CRITICAL QUESTIONS:
REFERENCE:

Attendance Observation Performance Record Pretest Critical Viva Total


10 4 4 Maintenance 4 Thinking 10 40
4 4

SIGNATURE OF THE TEACHER

Department of Pharmaceutical Technology, JIS University


EXPERIMENT NO. - 3

SERIAL No: DATE:

SIMPLE STAINING

AIM / OBJECTIVE

To perform simple staining.

PRINCIPLE: SIMPLE STAINING

AIM: To perform simple staining.

PRINCIPLE:

The simple stain can be used as a quick and easy way to determine cell shape, size and arrangements of
bacteria. True to its name, the simple stain is a very simple staining procedure involving single solution
of stain. Any basic dye such as methylene blue, safranin, or crystal violet can be used to color the
bacterial cells. These stains will readily give up a hydroxide ion or accept a hydrogen ion, which leaves
the stain positively charged. Since the surface of most bacterial cells and cytoplasm is negatively
charged, these positively charged stains adhere readily to the cell surface. After staining, bacterial cell
morphology (shape and arrangements) can be appreciated).

MATERIALS REQUIRED:
GLASSWARES:
Glass slide
Inoculation needle for stab culture technique
INSTRUMENTS REQUIRED:
Laminar air flow
Burners
Wire loops
Lighter/Match sticks
CHEMICALS REQUIRED:
Growth media for microbes
70% ethanol

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Methylene blue
Distilled water
Iodine solution

Simple Staining Procedure

PROCEDURE:
HEAT FIX SMEAR PREPARATION:
1. Using a sterilized inoculating loop, transfer loopful of liquid suspension containing bacteria to a slide
(clean grease free microscopic slide) or transfer an isolated colony from a culture plate to a slide with
a water drop.
2. Disperse the bacteria on the loop in the drop of water on the slide and spread the drop over an area
the size of a dime. It should be a thin, even smear.
3. Allow the smear to dry thoroughly.
4. Heat-fix the smear cautiously by passing the underside of the slide through the burner flame two or
three times. It fixes the cell in the slide. Do not overheat the slide as it will distort the bacterial cells.
STAINING:
1. Cover the smear with methylene blue and allow the dye to remain in the smear for approximately one
minute (Staining time is not critical here; somewhere between 30 seconds to 2 minutes should give
you an acceptable stain, the longer you leave the dye in it, the darker will be the stain).
2. Using distilled water wash bottle, gently wash off the excess methylene blue from the slide by
directing a gentle stream of water over the surface of the slide.
3. Wash off any stain that got on the bottom of the slide as well.
4. Saturate the smear again but this time with Iodine. Iodine will set the stain

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5. Wash of any excess iodine with gently running tap water. Rinse thoroughly. (You may not get
mention about step 4 and 5 in some text books)
6. Wipe the back of the slide and blot the stained surface with bibulous paper or with a paper towel.
7. Place the stained smear on the microscope stage smear side up and focus the smear using the 10X
objective.
8. Choose an area of the smear in which the cells are well spread in a monolayer. Center the area to be
studied, apply immersion oil directly to the smear, and focus the smear under oil with the 100X
objective.

Left: Cocci in Cluster; Right: Bacilli (Image source: microrao.com)

RESULTS:
The bacterial cells usually stain uniformly and the color of the cell depends on the type of dye used. If
methyene blue is used, some granules in the interior of the cells of some bacteria may appear more
deeply stained than the rest of the cell, which is due to presence of different chemical substances.

CRITICAL QUESTIONS:

REFERENCE:

Attendance Observation Performance Record Pretest Critical Viva Total


10 4 4 Maintenance 4 Thinking 10 40
4 4

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SIGNATURE OF THE TEACHER

EXPERIMENT NO. - 4

SERIAL No: DATE:

GRAM STAINING

AIM / OBJECTIVE

To perform gram staining.

PRINCIPLE:
.
It is one of the differential stains that are used to characterize bacteria in one of two groups: either gram
positive bacteria or gram negative bacteria.

Gram positive bacteria will typically have a stronger affinity for crystal violet on applying gram's iodine
than the gram negative cell wall.

Being a mordant, gram's iodine forms a complex with crystal violet in the stain that has attached more
tightly to the cell wall of gram positive bacteria than that of the gram negative bacteria.

Whereas the gram positive bacteria stain violet as a result of the presence of a thick peptidoglycan layer
in the walls of their cell, the gram negative bacteria stain red, due to the thinner peptidoglycan layer in
their cell wall (a thicker peptidoglycan layer allows for the retention of the stain, but a thinner layer does
not).

The staining involves 3 major steps/processes that include;

 Staining with crystal violet (a water soluble dye),


 De-colorization (using ethanol/acetone),
 Counterstaining (using Safranin),

Due to the differences in the thickness of the peptidoglycan layer on the cell walls of these bacteria,
gram positive bacteria will retain the crystal violet stain after the de-colorization process using ethyl
alcohol/acetone.

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After staining the sample with crystal violet, ethyl alcohol is used to decolorize the sample. It achieves
its purpose by dehydrating the peptidoglycan layer by tightening and shrinking it. In doing so, large
crystal violet cannot penetrate the tightened layer of peptidoglycan, and hence it is trapped in the cell
wall of gram positive bacteria.

On the other hand, the outer membrane of the gram negative cells cannot retain the crystal violet iodine
complex and hence the color is lost.

Safranin is a lighter stain as compared to crystal violet and thus it does disrupt the purple coloration in
the gram positive cells.

In an aqueous solution, crystal violet dissociates into ions of CV+ and CV-. These ions penetrate the
walls and membranes of both gram positive and negative cells.

CV+ will interact with the negatively charged components of the bacterial cells, and take up the purple
coloration. On adding iodine, iodine cations (I- or I3-) interact with CV+, which results in the formation
of larger complexes of CVI within the cytoplasm and the outer layers of the cell.

On adding the decolorizing agent (ethanol), it interacts with the membrane lipids of both the gram
positive and gram negative positive and gram negative.

This results in the loss of the outer membrane, which in turn leaves the peptidoglycan layer exposed. For
the gram negative cells, ethanol causes the walls to be leaky and hence they cannot hold the large
complexes of CV-L during de-colorization.

In some of the staining processes using gram stain, a pattern of gram-variables are obtained, which is a
mix of pink and purple.

Some generas, such as Arthrobacter, Actinomyces and Corynebacterium have a cell wall that is
particularly sensitive to breakage during cell division.

This results in gram negative staining of the gram positive cells.

On the other hand in cultures of Clostridium and Bacillus, the reduced thickness of peptidoglycan during
growth coincides with an increased number of cells that in turn stain gram negative.

MATERIALS REQUIRED:

GLASSWARES:
Glass slide
Inoculation needle for stab culture technique

INSTRUMENTS REQUIRED:
Laminar air flow
Burners
Wire loops

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Lighter/Match sticks

CHEMICALS REQUIRED:
Growth media for microbes
70% ethanol
Methylene blue
Distilled water
Gram’s Iodine solution
Safranine

PROCEDURE:

SMEAR PREPARATION:
It is important to note that the thickness of the sample smear on the slide is an important consideration
during the preparation of the sample. The smear should not be too think or too thin.

Label the slide;

Bacteria- smear the sample on the slide using an inoculating needle. This can also be done by
introducing a drop of saline on the slide followed by the sample and then mixing.

This should then be left to air dry before heat fixing by carefully passing the slide through the Bunsen
burner (avoid burning the sample).

Actinomycetes- same as bacteria, but by trying to get a portion of the colony on the slide while it is still
intact, this can be achieved by using a scalpel,

STAINING PROCEDURE:

Flood the slide with crystal violet staining reagent for about 1 minute ,
Wash the slide using a gentle, indirect stream of tap water for about 2 seconds, flood the slide
with a mordant (Gram’s iodine) then wait for a minute,
 Wash the slide for the again in a gentle, indirect stream of tap water for about 2 seconds,
 Flood the slide with the decolorizing agent then wait for 15 seconds. This can also be done by
adding a drop by drop to the slide until the decolorizing agent running from the slides runs
clear,
 Flood the slide using counterstain safranin( and wait for about a minute (30 seconds to 1
minute)
 Wash the slide using a gentle and indirect stream of tap water to a point where the color
appears in the effluent and then blot dry the absorbent paper,
 Add a drop of immersion oil on the stained sample and observe under the microscope.

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OBSERVATION:
Pink coloured colonies have been observed under microscope.

RESULT:
From the above observation we can conclude that those colonies obtain gram negative bacteria.

CRITICAL QUESTIONS:

REFERENCE:

Attendance Observation Performance Record Pretest Critical Viva Total


10 4 4 Maintenance 4 Thinking 10 40
4 4

SIGNATURE OF THE TEACHER

Department of Pharmaceutical Technology, JIS University


EXPERIMENT NO. - 5

SERIAL No: DATE:

ACID FAST STAINING

AIM / OBJECTIVE

To perform acid fast staining.

PRINCIPLE:
The main aim of this staining is to differentiate bacteria into acid fast group and non-acid fast groups.
This method is used for those microorganisms which are not staining by simple or Gram staining
method, particularly the member of genus Mycobacterium, are resistant and can only be visualized by
acid-fast staining.

When the smear is stained with carbol fuchsin, it solubilizes the lipoidal material present in the
Mycobacterial cell wall but by the application of heat, carbol fuchsin further penetrates through lipoidal
wall and enters into cytoplasm. Then after all cell appears red. Then the smear is decolorized with
decolorizing agent (3% HCL in 95% alcohol) but the acid fast cells are resistant due to the presence of
large amount of lipoidal material in their cell wall which prevents the penetration of decolorizing
solution. The non-acid fast organism lack the lipoidal material in their cell wall due to which they are
easily decolorized, leaving the cells colorless. Then the smear is stained with counterstain, methylene
blue. Only decolorized cells absorb the counter stain and take its color and appear. MATERIALS
REQUIRED:

GLASSWARES:
Glass slide
Inoculation needle for stab culture technique

INSTRUMENTS REQUIRED:
Laminar air flow
Burners
Wire loops
Lighter/Match sticks

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CHEMICALS REQUIRED:
Growth media for microbes
70% ethanol
Carbol fuschin
Distilled water
Malachite green
PROCEDURE:

Prepare bacterial smear on clean and grease free slide, using sterile technique.

Allow smear to air dry and then heat fix.

Alcohol-fixation: This is recommended when the smear has not been prepared from sodium hypochlorite
(bleach) treated sputum and will not be stained immediately. M. tuberculosis is killed by bleach and
during the staining process. Heat-fixation of untreated sputum will not kill M. tuberculosis whereas
alcohol-fixation is bactericidal.

Cover the smear with carbol fuchsin stain.

Heat the stain until vapour just begins to rise (i.e. about 60 C). Do not overheat. Allow the heated stain
to remain on the slide for 5 minutes.

Heating the stain: Great care must be taken when heating the carbol fuchsin especially if staining is
carried out over a tray or other container in which highly flammable chemicals have collected from
previous staining. Only a small flame should be applied under the slides using an ignited swab
previously dampened with a few drops of acid alcohol or 70% v/v ethanol or methanol. Do not use a
large ethanol soaked swab because this is a fire risk.

Wash off the stain with clean water.

Note: When the tap water is not clean, wash the smear with filtered water or clean boiled rainwater.

Cover the smear with 3% v/v acid alcohol for 5 minutes or until the smear is sufficiently decolorized, i.e.
pale pink.

Caution: Acid alcohol is flammable, therefore use it with care well away from an open flame.

Wash well with clean water.

Cover the smear with malachite green stain for 1–2 minutes, using the longer time when the smear is
thin.

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Wash off the stain with clean water.

Wipe the back of the slide clean, and place it in a draining rack for the smear to air-dry (do not blot dry).

OBSERVATION: Red coloured rods are visible.

RESULT: The sample contains Mycobacterium sp.

CRITICAL QUESTIONS:

REFERENCE:

Attendance Observation Performance Record Pretest Critical Viva Total


10 4 4 Maintenance 4 Thinking 10 40
4 4

SIGNATURE OF THE TEACHER

Department of Pharmaceutical Technology, JIS University


EXPERIMENT NO. - 6

SERIAL No: DATE:

ISOLATION OF PURE CULTURE OF MICROORGANISMS BY MULTIPLE STREAK


PLATE TECHNIQUE AND OTHER TECHNIQUES

AIM / OBJECTIVE

To perform isolation of microorganisms.

PRINCIPLE:
The streak plate method is a rapid qualitative isolation method. The techniques commonly used for
isolation of discrete colonies initially require that the number of organisms in the inoculums be reduced.
It is essentially a dilution technique that involves spreading a loopful of culture over the surface of an
agar plate. The resulting diminution of the population size ensures that, following inoculation, individual
cells will be sufficiently far apart on the surface of the agar medium to effect a separation of the
different species present. Although many type of procedures are performed, the four ways or quadrant
streak is mostly done.
MATERIALS REQUIRED:

1. Mixed culture of bacteria.


2. Sterile petri dish with appropriate bacterial media(such as trypticase soy agar, nutrient agar).
3. Inoculating loop (usually nichrome, a nickel-chromium alloy, or platinum; it may also be a single-use
disposable plastic loop, which would be discarded between sectors rather than resterilized).
4. Bunsen burner.
5. Marking pen.

PROCEDURE:

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All the process is done in a laminar air flow cabinet aseptically.

LABEL A PETRIDISH:

Petri dishes are labelled on the bottom rather than on the lid. Write close to the edge of the bottom of the
plate to preserve area to observe the plate after it has incubated. Labels usually include the organism
name, type of agar, date, and the plater's name or initials. Using sterile cotton swabs, remove any visible
water on the agar in the plate or around the inner rim of the petri plate. Observe the plate and mentally
divide it into three sectors. The plate will then be turned clockwise (if you are right handed) with the
agar side up. The second sector will then be at the top for streaking and then the plate is turned again so
that the third sector can be streaked.

STERILIZE THE TRANSFER LOOP BEFORE OBTAINING A SPECIMEN:

To streak a specimen from a culture tube, metal transfer loops are first sterilized by flaming the wire
loop held in the light blue area of a Bunsen burner just above the tip of inner flame of the flame until it
is red-hot. If a hot incinerator is available, the loop may be sterilized by holding it inside the incinerator
for 5 to 7 seconds. Once sterile, the loop is allowed to cool by holding it still. Do not wave it around to
cool it or blow on it. When manipulating bacteria, transfer loops are usually held like a pencil. If plastic
disposable loops are being utilized, they are removed from the packaging to avoid contamination and
after being used, are discarded into an appropriate container. A new loop is recommended for each
sector of an isolation streak plate.

OPEN THE CULTURE TUBE AND COLLECT ASAMPLE OF SPECIMEN USING THE
STERILE LOOP:

Isolation can be obtained from any of a variety of specimens. This protocol describes the use of a mixed
broth culture, where the culture contains several different bacterial species or strains. The specimen
streaked on a plate could come in a variety of forms, such as solid samples, liquid samples, and cotton or
foam swabs. Material containing possibly infectious agents should be handled appropriately in the lab
using bio safety procedure.
Remove the test tube cap. It is recommended that the cap be kept in your right hand (the hand holding
the sterile loop). Curl the little finger of your right hand around the cap to hold it or hold it between the
little finger and third finger from the back. Modern test tube caps extend over the top of the test tube,
keeping the rim of the test tube sterile while the rim of the cap has not been exposed to the bacteria. The
cap can also be placed on the disinfected table, if the test tube is held at an angle so that air
contamination does not fall down into the tube. Insert the loop into the culture tube and remove a loopful
of broth. Replace the cap of the test tube and put it back into the test tube rack.

STREAK THE PLATE:

The lid of the agar plate has to be opened just sufficiently enough to streak the plate with the inoculation
loop. Minimize the amount of agar and the length of time the agar is exposed to the environment during
the streak process.

THREE SECTOR STREAK (t streak):

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1. Sterilize the wire loop.
2. Cool the loop by touching it on the edge of the sterile agar plate.
3. Dip the loop into the broth culture containing the mixture of bacteria.
4. Lift the lid of the plate just enough to insert the loop. Drag the loop over the surface of the top one-third
of the plate back and forth in a "zig-zag" formation.
5. The loop has picked up thousands of bacteria which are spread out over the surface of the agar.
6. Sterilize the loop in the flame.
7. Turn the plate 90 degrees and drag the loop through the area you have just streaked two to three times
and continue to drag the loop in a "zig-zag" formation in the remaining half of the plate without
touching that area again.
8. Sterilize the loop in the flame.
9. Turn the plate 90 degrees. Repeat the procedure. Drag the loop two to three times through the area you
just streaked, and fill in the remaining area of the plate (zig-zag formation), being very careful not to
touch any of the areas you previously streaked.
10. Incubate the plate for 24 hours. If you streaked correctly, you will see isolated colonies in the third
sector. The heaviest growth will be in the first sector. There will be less growth and some isolated
colonies in the second sector. The third area should have the least growth with isolated colonies.

FOUR QUADRANT STREAK :

1. Loosen the cap of the bottle containing the inoculum.


2. Hold an inoculation loop in your right hand.
3. Flame the loop and allow it to cool.
4. Lift the test tube containing the inoculum with your left hand.
5. Remove the cap/ cotton wool plug of the test tube with the little finger of your right hand.
6. Flame the neck of the test tube.
7. Insert the loop into the culture broth and withdraw. At all times hold the loop as still as possible.
8. Flame the neck of the test tube again.
9. Replace the cap/ cotton wool plug of the test tube using the little finger of your right hand. Place the test
tube in a rack. For a liquid culture, dip the loop into the broth, or for solid media, lightly touch a colony
with the loop.
10. Partially lift the lid of the Petri dish containing the solid medium.
11. Place a loopful of the culture on the agar surface on the area 1. Flame the loop and cool it for 5 seconds
by touching an unused part of the agar surface close to the periphery of the plate, and then drag it rapidly
several times across the surface of area1.
12. Remove the loop and close the Petri dish.
13. Reflame and cool the loop, and turn the petri dish 90°C then touch the loop to a corner of the culture in
area1 and drag it several times across the agar in area 2, hitting the original streak a few times. The loop
should never enter area 1 again.
14. Remove the loop and close the Petri dish.
15. Reflame and cool the loop and again turn the dish 90°C anticlockwise. streak area 3 in the same manner
as area 2, hitting last area several times.
16. Remove the loop and close the Petri dish.
17. Flame the loop, again turn the dish 90°C and then drag the culture from a corner of a area3 across area 4,
contacting area 3 several times and drag out the culture as illustrated. Using a wider streak. Do not let

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the loop touch any of the previously streaked areas. The flaming of the loop at the points indicated is to
effect the dilution of the culture so that fewer organisms are streaked in each area, resulting in the final
desired separation.
18. Remove the loop and close the Petri dish.
19. Tape the plate closed and incubates the plate in an inverted position in an incubator for 24-48 hours.
20. Flame the loop before putting it aside.
OBSERVATION:

Fig 1: Staphylococcus aureus colony on nutrient agar


1. Area of initial inoculation and the first streak yields heavy growth.
2. Area of the second streak from the area 1 yields gives dense growth.
3. Area of the third streak from the area2 yields weak growth.
4. Area of the fourth streak from the area3 yields single colonies.

RESULT:
Isolation of microorganisms has done successfully.

CRITICAL QUESTIONS:

REFERENCE:

Attendance Observation Performance Record Pretest Critical Viva Total


10 4 4 Maintenance 4 Thinking 10 40
4 4

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SIGNATURE OF THE TEACHER

EXPERIMENT NO. - 7

SERIAL No: DATE:

MOTILITY DETERMINATION BY HANGING DROP METHOD

AIM / OBJECTIVE

To determine the motility of microorganisms.

PRINCIPAL:

Hanging drop preparation is a special type of wet mount (in which a drop of medium containing the
organisms is placed on a microscope slide), often is used in dark illumination to observe the motility of
bacteria.In this method a drop of culture is placed on a coverslip that is encircled with petroleum jelly
(or any other sticky material). The coverslip and drop are then inverted over the well of a depression
slide. The drop hangs from the coverslip, and the petroleum jelly forms a seal that prevents evaporation.
This preparation gives good views of microbial motility.

HANGING DROP METHOD

MATERIALS REQUIRED:
1. Glass slides (glass slide with depression) or Normal glass slide with adhesive or paraffin ring
2. Paraffin wax
3. Loop
4. Coverslip
5. Microscope
6. Bunsen burner

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7. Young broth culture of motile bacteria

PROCEDURE:
1. Take a clean glass slide and apply paraffin ring, adhesive tape ring to make circular concavity. (This
step is not needed if a glass slide with depression is available).
2. Hold a clean coverslip by its edges and carefully dab Vaseline on its corners using a toothpick.
3. Place a loopful of the broth culture to be tested in the center of the prepared coverslip.
4. Turn the prepared glass slide or concavity slide upside down (concavity down) over the drop on the
coverslip so that the vaseline seals the coverslip to the slide around the concavity.
5. Turn the slide over so the coverslip is on top and the drop can be observed hanging from the
coverslip over the concavity.
6. Place the preparation in the microscope slide holder and align it using the naked eye so an edge of the
drop is under the low power objectives.
7. Turn the objective to its lowest position using the coarse adjustment and CLOSE THE
DIAPHRAGM.
8. Look through the eyepiece and raise the objective slowly using the coarse adjustment knob until the
edge of the drop is observed as an irregular line crossing the field.
9. Move the slide to make that line (the edge of the drop) pass through the center of the field.
10. Without raising or lowering the tube, swing the high dry objective into position (Be sure the high dry
objective is clean).
11. Observe the slide through the eyepiece and adjust the fine adjustment until the edge of the drop can
be seen as a thick, usually dark line.
12. Focus the edge of the drop carefully and look at each side of that line for very small objects that are
the bacteria. The cells will look either like dark or slightly greenish, very small rods or
spheres. Remember the high dry objective magnifies a little less than half as much as the oil
immersion objective.
13. Adjust the light using the diaphragm lever to maximize the visibility of the cells.
14. Observe the cells noting their morphology and grouping and determine whether true motility can be
observed.
15. Brownian movement should be visible on slides of all the organisms, but there should also show true
motility.
16. Wash the depression slide and after soaking in lysol buckets or discard the prepared glass slide.

OBSERVATION:
Motility of bacteria has been observed.
RESULT:
The hanging drop method has been successfully performed.
CRITICAL QUESTIONS:
REFERENCE:
Attendance Observation Performance Record Pretest Critical Viva Total
10 4 4 Maintenance 4 Thinking 10 40
4 4

Department of Pharmaceutical Technology, JIS University


SIGNATURE OF THE TEACHER

EXPERIMENT NO. - 8

SERIAL No: DATE:

BACTEROIOLOGICAL ANALYSIS OF WATER

AIM / OBJECTIVE

To perform bacteriologicalal analysis of water.

PRINCIPLE:
There will be three principal tests: the presumptive, confirmed and completed tests.

The Presumptive Test In the presumptive test, a series of lactose broth tubes are inoculated with
measured amounts of the water sample to be tested. The series of tubes may consist of three or four
groups of three, five or more tubes. The more tubes utilized, the more sensitive the test. Gas production
in any one of the tubes is presumptive evidence of the presence of coliforms. The most probable number
(MPN) of coliforms in 100 ml of the water sample can be estimated by the number of positive tubes (see
MPN Table).

The Confirmed Test If any of the tubes inoculated with the water sample produce gas, the water is
presumed to be unsafe. However, it is possible that the formation of gas may not be due to the presence
of coliforms. In order the confirm the presence of coliforms, it is necessary to inoculate EMB (eosin
methylene blue) agar plates from a positive presumptive tube. The methylene blue in EMB agar inhibits
Grampositive organisms and allows the Gram-negative coliforms to grow. Coliforms produce colonies
with dark centers. E. coli and E. aerogenes can be distinguished from one another by the size and color
of the colonies. E. coli colonies are small and have a green metallic sheen, whereas E. aerogenes forms

Department of Pharmaceutical Technology, JIS University


large pinkish colonies. If only E. coli or if both E. coli and E. aerogenes appear on the EMB plate, the
test is considered positive. If only E. aerogenes appears on the EMB plate, the test is considered
negative. The reasons for these interpretations are that, as previously stated, E. coli is an indicator of
fecal contamination, since it is not normally found in water or soil, whereas E. aerogenes is widely
distributed in nature outside of the intestinal tract.

The completed test is made using the organisms which grew on the confirmed test media. These
organisms are used to inoculate a nutrient agar slant and a tube of lactose broth. After 24 hours at 37°C,
the lactose broth is checked for the production of gas, and a Gram stain is made from organisms on the
nutrient agar slant. If the organism is a Gram-negative, nonspore-forming rod and produces gas in the
lactose tube, then it is positive that coliforms are present in the water sample.

FIRST PERIOD

MATERIALS REQUIRED:

1. Nine tubes of double-strength lactose broth

2. 10, 1.0 and 0.1 ml pipets

3. Water samples

PROCEDURE: (work in groups of four)

PRESUMPTIVE TEST

Take a water sample (dilute as instructed in some cases) and inoculate three tubes of lactose broth with
10 ml, three tubes with 1.0 ml and three tubes with 0.1 ml. 2. Incubate all tubes at 37o C for 24 hours.

OBSERVATION:

RESULT:

SECOND PERIOD

MATERIAL REQUIRED:

EMB agar plates

PROCEDURE:

PRESUMPTIVE TEST:

Observe the number of tubes at each dilution that show gas production in 24 hrs. Record results. 2.
Reincubate for an additional 24 hours at 37°C.

CONFIRMED TEST:

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Inoculate an EMB plate with material from a tube containing gas. 2. Invert and incubate the plate at
37°C for 24 hours.

OBSERVATION:

RESULT:

THIRD PERIOD

MATERIALS REQUIRED:

1. Lactose broth tubes

2. Nutrient agar slants

PROCEDURE:

PRESUMPTIVE TEST

Observe the number of tubes at each dilution that show gas. Record results and determine the most
probable number index.

CONFIRMED TEST

1. Observe EMB agar plates. A positive confirmed test is indicated by small colonies with dark centers
and a green metallic sheen (E. coli).

OBSERVATION:

RESULT:

COMPLETED TEST

Inoculate a lactose broth tube and a nutrient agar slant with organisms from the EMB plate. 2. Incubate
the broth tube and agar slant at 37°C for 24 hours.

FOURTH PERIOD

PROCEDURE:

COMPLETED TEST:

Check for gas production in the lactose broth tube. 2. Make a Gram stain from the organisms on the
nutrient agar slant.

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OBSERVATION:

RESULT:

CRITICAL QUESTIONS:

REFERENCE:
Attendance Observation Performance Record Pretest Critical Viva Total
10 4 4 Maintenance 4 Thinking 10 40
4 4

SIGNATURE OF THE TEACHER

Department of Pharmaceutical Technology, JIS University


EXPERIMENT NO. - 9

SERIAL No: DATE:

IMVIC TEST

AIM / OBJECTIVE

To perform imvic test of water.

PRINCIPLE:

Each of the letters in “IMViC” stands for one of these tests. “I” is for indole; “M” is for methyl red; “V”
is for Voges-Proskauer, and “C” is for citrate, lowercase “i” is added for the ease of
pronunciation. IMViC is an acronym that stands for four different tests
 Indole test
 Methyl red test
 Voges-Proskauer test
 Citrate utilization test
To obtain the results of these four tests, three test tubes are inoculated: tryptone broth (indole test),
methyl red – Voges Proskauer broth (MR-VP broth), and citrate. IMViC tests are employed in the
identification/differentiation of members of family enterobacteriaceae.

MATERIALS REQUIRED:
Sulfite indole motility medium
Kovac’s reagent
Methyl red Vogas Proskauer broth
Methyl red reagent
Barritt’s reagent A

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Barritt’s reagent B
Simmon’s citrate agar

PROCEDURE:

INDOLE TEST:

It is performed on sulfide-indole-motility (SIM) medium or in tryptophan broth, or in motility urease


indole (MIU) medium. Result is read after adding Kovac’s reagent.

OBSERVATION:

1. The positive result is indicated by the red layer at the top of the tube after the addition of Kovács
reagent.
2. A negative result is indicated by the lack of color change at the top of the tube after the addition of
Kovács reagent.

RESULT:
Positive-development of Red-ring

Methyl red test and Voges-Proskauer test both are done in methyl red–Voges-Proskauer (MR-VP)
broth, but the reagents that are added varies according to the test.

METHYL RED TEST:

OBSERVATION AND RESULT:


 Positive methyl red test are indicated by the development of red color after the addition of methyl red
reagent.
 A negative methyl red test is indicated by no color change after the addition of methyl red reagent
VOGAS PROSKAUER TEST:

OBSERVATION AND RESULT:


1. Negative test is indicated by lack of color change after the addition of Barritt’s A and Barritt’s B
reagents.
2. A positive Voges-Proskauer test is indicated by the development of red-brown color after the
addition of Barritt’s A and Barritt’s B reagents.
3.
CITRATE UTILIZATION TEST:

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The test is performed on Simmons citrate agar.

OBSERVATION AND RESULT:


1. Negative citrate utilization test is indicated by the lack of growth and color change in the tube
2. A positive citrate result as indicated by growth and a blue color change.

CRITICAL QUESTIONS:

REFERENCE:

Attendance Observation Performance Record Pretest Critical Viva Total


10 4 4 Maintenance 4 Thinking 10 40
4 4

SIGNATURE OF THE TEACHER

Department of Pharmaceutical Technology, JIS University


Department of Pharmaceutical Technology, JIS University