INDEX

1. BASIC LABORATORY ETHICS AND SAFETY MEASUREMENTS

2. STUDY OF BASIC LAB EQUIPMENT

3. STUDY OF MICROSCOPE

4. PREPARATION OF GROWTH MEDIA

5. STUDY OF MICROBIOLOGY OF AIR

6. ISOLATION OF BACTERIAL CELL BY STREAK PLATE METHOD

7. PREPARATION OF SMEAR FROM GIVEN MEDIA

8. SIMPLE STAINING OF GIVEN CULTURE

9. GRAM STAINING OF GIVEN CULTURE

10. STUDY OF MICROBIOLOGY OF WATER

11. STUDY OF MICROBIOLOGY OF SOIL

12. STUDY OF MACCONKEY AGAR MEDIA

13. IDENTIFICATION AND MICROSCOPIC STUDIES OF FUNGI

14. STUDY OF FUNGI BY SIMPLE STAINING METHOD

15. STUDY OF STERILIZATION TECHNIQUE

 EXPERIMENT NO.1
BASIC LABORATORY ETHICS AND SAFETY MEASUREMENTS

Most Microbiology laboratories require the use of living pathogenic microorganisms, such
microorganisms must be handled with extreme care to avoid chances of infection and
contamination for this reason we use aseptic techniques which are an important part of safety in
labs. The following basic steps should be taken to reduce chances of contamination and control
on microbial contamination.
1. Before working in lab place your belongings at their defined place, don’t place them on working
desk.
2. Minimize all air movements, close the door, windows and electric fans while transferring the
culture media.
3. Before and after working clean your working desk with ethyl alcohol or other suitable
disinfectants.
4. Sterilize all needle, loops over the flame, and sterilize your glassware in hot air oven before and
after your experiments.
5. Don’t place contaminated instruments such as inoculating loops, needles, pipettes and other
glassware in your inventory rather sterilize them and store.
6. Do not place prepared cultures un attended. In case of spill, spilled cultures must not be touched
with unprotected hands, wearing disposable gloves, disinfect the area by covering the spill with
several layers of paper towel/cloth soaked in a suitable disinfectant and leave for 15-30 minutes.
7. Autoclave discarded cultures.
8. All incubated cultures must be labeled properly with name and date.
9. Turn Bunsen burner off when not in use. Check your burner before leaving lab.
10. Wearing lab coat is necessary, gloves and eye protected glasses should be used while working.
11. Do not touch your skin, eye or face while working in lab.
12. Wash your hands thoroughly while leaving lab.
13. Make sure you do not leave any mess behind on your work desk.
 EXPERIMENT NO. 2
STUDY OF BASIC LAB EQUIPMENT
A culture containing a single, unadulterated species of cells is called Pure culture. To isolate and study
microbes in pure culture the microbiologists require basic lab apparatus and applications for specific
techniques.

1. Media;
The survival and continues growth of microbes depends on adequate supply of environment favorable for
growth and nutrients for survival.
A solution containing these nutrient called Culture Media.
The microbes that grow and multiply in or on a culture medium are referred to as a culture.
Microbes that are introduced into a culture medium to initiate growth are called an inoculum.
i. Broth Media; A liquid that lacks solidifying agent.
ii. Agar Media; A broth medium supplemented by solidifying agent.
iii. Semi Solid Medium; A concentration of less than 1% agar result in semi solid media.
iv. Agar Slant; it in liquefied state, solid media can be placed in a test tube which is then allowed to cool
and harden in a slanted position producing agar slant.
v. Agar Deep Tube; Tube that following preparation are allowed to
harden in upright position called agar deep tube.
vi. Agar Plate; Agar slant or agar deep tubes may be liquefied in a boiling water bath and poured into petri
dishes producing agar plate.

2. Culture tubes and Petri Dishes; Glass test tubes and glass or petri dishes
are used to culture microorganisms. Petri dishes provide longer surface
area for growth and cultivation. Petri dishes are manufactured in various sizes for routine purpose approx.
15cm in diameter.

3. Transfer Instruments;
Microorganisms must be transformed from one vessel to another for study called sub-culturing.

4. Wire Loops and Needles;

Wire loops and needles are made from inert metals. They are extremely durable instruments and easily
sterilized by incineration or flame heat.

5. Pipette;
A pipette is another instrument used for aseptic transfer. They are smaller in size and their function is to draw
liquids from different vessels and transfer from one to another. They are calibrated to deliver different
volumes.

6. Cultivated Chambers;
To provide optimum temperature to microorganism. Eg; Incubators, Oven and water bath are used to cultivate
microorganisms.

7. Refrigerator;
A refrigerator is used for maintenance and storage of stock cultures between sub-culturing periods and storage
of sterile media to prevent dehydration.
 8. Autoclave;
Autoclave is used to sterilize the media after is preparation. Autoclave is a tough double walled chamber in
which air is replaced by pure saturated steam under pressure.

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Nutrient Growth Media

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Autoclave

Hot Air Oven

 EXPERIMENT NO. 3
STUDY OF MICROSCOPE
A device which is used as scientific instrument for extending the visibility of micro-organism for human eye. In
order to advance study minute things (living of non-living) for detailed structural study.
It has the ability to enhance image by x10 and x1000.
Parts of Microscope:
Basic important parts of microscope;
1) Eye piece 5) Object
2) Body tube 6) Stages
3) Coarse and fine knobs 7) Diaphragm
4) Nose piece 8) Light sources

1) Eye piece:
It is a pair of lense through which observer looks at the image of specimen. The lense increase the
magnification which shows the specimen easily.
5x, 10x, 15x, 20x ……100x…... 1000x etc.
2) Body Tube:
It is a long tube of microscope. It forms the body. It has ocular part as the upper part of the revolving
nose piece.
3) Coarse and Fine Knobs:
These are two fine knobs at the either sides of the arm which move the stage. Which helps in building
the focus.
Coarse knobs are used for rough focus, and fine knobs are used for fine focus.
4) Nose Piece:
It’s the place were objectives / lenses are placed.
5) Objectives:
These are the lenses of different power, their power can vary from 4x to 400x.
6) Stage: It is the horizontal square metallic plate, on which glass slides are placed. There are clips to hold the
glass slide above the light source. A hole is present in stage which allows light to pass through the slide and
help form the image of the specimen.
7) Diaphragm:
It is a circular disc with different sizes of holes in it allow the light control, it has different prism fixed in
it. It usually blue slip in it fixed which gives color visibility to different specimen.
8) Light source:
It provides the light, usually a white or yellow blub is present in microscopes. There is a side button
which controls the intensity of the light.
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Experiment No.3
Study of Microscope
EXPERIMENT NO. 4
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Nutrient Broth Media

Nutrient Agar Media

 EXPERIMENT NO. 5
MICROBIOLOGY OF AIR
Apparatus:
Petri dishes
Beakers
Stirrer
Conical flasks
Glass slide
Wire loop

Chemicals:
Nutrient Agar media 2.8g
Distilled water 100ml
Safranin dye
Crystal violet dye
Decolorizer

Theory:
Different types of microbes are present in air, we will observe different types of bacteria by gram
staning, under microscope after cultivating bacteria in petri dishes containing nutrient agar media.

Procedure:
 Weigh nutrient agar media and dissolve in distilled water, pour is carefully in petri dishes under
aseptic conditions.
 Always use sterile petri dishes.
 Pour nutrient agar media in all petri dishes equally.
 After an hour the liquid will be solidified.
 Label all nutrient agar media prepared 4 petri dishes as A, B, C, and D.
 Petri dish A is taken as a controlled standard. And will remain covered
 Now give air exposure to all petri dishes except A for different time duration.
 Give air exposure to petri dish B for 10 mins, Petri dish C for 15mins, Petri dish D for 20 mins
and cover then.
 Place all 4 petri dishes, A, B, C, and D in an incubator for bacterial growth to take place.
 After 24 hours of incubation you will observe the bacterial colonies.
 Physical state of bacterial colonies are determined by their color, size etc.
 Take bacterial culture on the slides and observe under microscope for identification of bacterial
specie.
Precaution:
All performance must be done in sterile conditions.
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 EXPERIMENT NO. 6
ISOLATION OF BACTERIAL CELLS BY STREAK PLATE METHOD

THEORY
One bacterial cell will create a colony as it multiplies. The streak process is intended to create a region
where the bacteria are so dilute that when each bacterium touches the surface of the agar, it is far enough
away from other cells so that an isolated colony can develop. In this manner, spreading an inoculum with
multiple organisms will result in isolation of the different organisms.

PRINCIPLE OF STREAKING
The inoculum is diluted by streaking it across the surface of the agar plate. While streaking in successive
areas of the plate, the inoculum is diluted to the point where there is only one bacterial cell deposited every
few millimeters on the surface of the agar plate. When these lone bacterial cells divide and give rise to
thousands and thousands of new bacterial cells, an isolated colony is formed. Pure cultures can be obtained
by picking well-isolated colonies and re-streaking these on fresh agar plates.

PURPOSE OF STREAKING

To produce isolated colonies of an organism (mostly bacteria) on an agar plate. This is useful when
we need to separate organisms in a mixed culture (to purify/isolate a particular strain from contaminants)
or when we need to study the colony morphology of an organism.

Materials required
A source of bacteria (stock culture, previously streaked agar plate or any other inoculum)
Inoculation loop
Bunsen burner
Agar plate (nutrient agar or any other agar medium)
Petri dishes

PROCEDURE
 Sterilize the inoculating loop on the bunsen burner by putting the loop into the flame until it is red
hot. Allow it to cool.
 Pick an isolated colony from the agar plate culture and spread it over agar media, using close
parallel streaks or Insert your loop into the tube/culture bottle and remove some inoculum. You
don’t need a huge chunk.
  Immediately streak the inoculating loop very gently all over on petri plate in continues pattern
using a back and forth motion.

TIPS FOR THE BEST RESULTS

 Use only a small amount of inoculum.
 Streak lightly so that you do not gouge the agar.
 Flame the loop before and after you streak.
 Make sure the surface of the plate is free of droplets of condensed moisture.

RESULTS

The streaked plate is incubated at 37°C for 24 hours. Examine the colonies grown on the plate
carefully. All colonies should have the same general appearance. If there is more than one type of
colony, each type should be streaked again on a separate plate to obtain a pure culture.
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Continuous Streak Method

 EXPERIMENT NO. 7
SMEAR PREPARATION FROM PLATE OR BROTH
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 EXPERIMENT NO. 8
SIMPLE STAINING OF BACTERIA

Cultures: given culture of unknown bacterial species.

Simple Staining by Methylene Blue, Staphylococcus Aureus
EXPERIMENT NO. 9
Gram Positive Purple Stain Gram Negative Pink Stain
 EXPERIMENT NO. 10
STUDY OF MICROBIOLOGY OF WATER
Apparatus:
Petri dishes
Beakers
Stirrer
Conical flasks
Glass slide
Wire loop

Chemicals:
Nutrient Agar media 2.8g
Distilled water 100ml
Safranin dye
Crystal violet dye
Decolorizer

Theory:
Microbiology of water is also aquatic microbiology. Water is good media for bacterial growth.

Procedure:
Weigh nutrient agar media and dissolve in distilled water, pour is carefully in petri dishes under aseptic
conditions.
Always use sterile petri dishes.
Pour nutrient agar media in all petri dishes equally.
After an hour the liquid will be solidified.
Label all nutrient agar media prepared 4 petri dishes as A, B, C, and D.
Petri dish A is taken as a controlled standard. And will remain covered
Take 4 test tubes and take 10ml sample water in test tube number 1 and take 9ml of distilled water in
test tube 2,3 and 4.
Now take 1ml sample water from test tube number 1 and transfer this 1ml water into test tube number 2,
take 1ml from test tube 2 and transfer in test tube 3 and so on so.
Now take 1ml from test tube 1, 2, 3 and 4 and pour it in petri dishes A, B, C and D respectively.
Cover petri dishes with lid and leave them for an hour.
Place all 4 petri dishes, A, B, C, and D in an incubator for bacterial growth to take place.
After 24 hours of incubation you will observe the bacterial colonies.
Physical state of bacterial colonies are determined by their color, size etc.
Take bacterial culture on the slides and observe under microscope for identification of bacterial specie.
Precaution:
All performance must be done in sterile conditions.

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Petri Dish A (Controlled) Petri Dish B (Dilution

1:10)

Petri Dish C (Dilution 1:100) Petri Dish D (Dilution 1:1000)

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Observation and Calculations

Obs. Petri Dilution Control No. Of Corrected No. of Colonies/ml

Dish Colonies Colonies
1. A Controlled 1 1-1=0 0

2. B 1:10 21 21-1= 20 20 x 10 = 200

3. C 1:100 10 10-1= 9 9 x 100 = 900

4. D 1:1000 8 8-1= 7 7 x 1000 = 7000

Total number of colonies = 8100

Average = 8100 / 3 = 2700
Characteristics of Observed colonies:
Color: slightly green white Transparency: opaque
Shape: Oval Margins: entire

Result:
The colonies are greeny white, opaque round with entire margin. Microscopic examination
shows that these are gram positive bacillus bacteria.
 EXPERIMENT NO. 11
STUDY OF MICROBIOLOGY OF SOIL
Apparatus:
Four Test Tubes
Four Petri Dish
Burner
Sterilized Petri Dish
Glass slide
Wire loop

Chemicals:
Nutrient Agar media 2.8g
Distilled water 100ml
Pinch of soil

For Gram Staining

Safranin dye
Crystal violet dye
Decolorizer

Theory:
Soil has been identified as the region of earth where geology and biology meet from
functional point of view. The soil may be considered as the land of surface, of earth crust
which provides substances for plants and animal life. It consist of mineral particles, organic
residues, water, gases like oxygen/nitrogen/carbon dioxide and microbial flora of soil.

Procedure:
Weigh nutrient agar media and dissolve in distilled water, pour is carefully in petri dishes
under aseptic conditions.
Always use sterile petri dishes.
Pour nutrient agar media in all petri dishes equally.
After an hour the liquid will be solidified.
Label all nutrient agar media prepared 4 petri dishes as A, B, C, and D.
Petri dish A is taken as a controlled standard. And will remain covered
Take 4 test tubes and mix pinch of soil in 10ml distilled water in test tube number 1
Now take 1ml soil water from test tube number 1 and transfer this 1ml water into test tube
number 2, take 1ml from test tube 2 and transfer in test tube 3 and so on so.
Now take 1ml from test tube 1, 2, 3 and 4 and pour it in petri dishes A, B, C and D
respectively.
Cover petri dishes with lid and leave them for an hour.
Place all 4 petri dishes, A, B, C, and D in an incubator for bacterial growth to take place.
After 24 hours of incubation you will observe the bacterial colonies.
Physical state of bacterial colonies are determined by their color, size etc.
Take bacterial culture on the slides and observe under microscope for identification of
bacterial specie.
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Petri Dish O (Controlled) Petri Dish A

Petri Dish B Petri Dish C

 Observation and Calculations

Obs. Petri Dilution Control No. Of Corrected No. of Colonies/ml

Dish Colonies Colonies
1. O Controlled 1 1-1=0 0

2. A 1:10 140 140-1= 139 139 x 10 = 1390

3. B 1:100 90 90-1= 89 89 x 100 = 8900

4. C 1:1000 50 50-1= 49 49 x 1000 = 49000

Total number of colonies = 59290

Average = 59290 / 3 = 19763
Characteristics of Observed colonies:
Color: Creamy white Transparency: opaque
Shape: Oval Margins: entire

Result:
water sample contain 3250 colonies per ml. Most of the bacteria found are bacilli, it was
proven in
 EXPERIMENT NO. 12
STUDY OF MACCONKEY AGAR MEDIA

THEORY:
MacConkey agar (MAC) was the first solid differential media to be formulated which was
developed at 20th century by Alfred Theodore MacConkey. MacConkey agar is a selective and
differential media used for the isolation and differentiation of non-fastidious gram-negative rods,
particularly members of the family Enterobacteriaceae and the genus Pseudomonas.

PRINCIPLE OF MACCONKEY AGAR

MacConkey agar is used for the isolation of gram-negative enteric bacteria and the
differentiation of lactose fermenting from lactose non-fermenting gram-negative
bacteria. Pancreatic digest of gelatin and peptones (meat and casein) provide the
essential nutrients, vitamins and nitrogenous factors required for growth of
microorganisms.
Lactose monohydrate is the fermentable source of carbohydrate. The selective
action of this medium is attributed to crystal violet and bile salts, which are inhibitory
to most species of gram-positive bacteria. Sodium chloride maintains the osmotic
balance in the medium. Neutral red is a pH indicator that turns red at a pH below 6.8
and is colorless at any pH greater than 6.8. Agar is the solidifying agent.

CHEMICALS:
 Peptone (Pancreatic digest of gelatin) 17 gm
 Proteose peptone (meat and casein) 3 gm
 Lactose monohydrate 10 gm
 Bile salts 1.5 gm
 Sodium chloride 5 gm
 Neutral red 0.03 gm
 Crystal Violet 0.001 g
 Agar 13.5 gm
 Distilled Water Add to make 1 Liter

PREPARATION OF MACCONKEY AGAR:

 1. Suspend 49.53 grams of dehydrated medium in 1000 ml purified/distilled
water.
2. Heat to boiling to dissolve the medium completely.
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
4. Cool to 45-50°C.
5. Mix well before pouring into sterile Petri plates.

RESULT INTERPRETATION ON MACCONKEY AGAR:

Lactose fermenting strains grow as red or pink and may be surrounded by a zone of
acid precipitated bile. The red color is due to production of acid from lactose,
absorption of neutral red and a subsequent color change of the dye when the pH of
medium falls below 6.8.

Lactose non-fermenting strains, such

as Shigella and Salmonella are colorless and transparent and typically do not alter
appearance of the medium. Yersinia enterocolitica may appear as small, non-lactose
fermenting colonies after incubation at room temperature.
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 EXPERIMENT NO. 13
IDENTIFICATION AND MICROSCOPIC STUDIES OF FUNGI

Definition:
Eukaryotic, heterotrophic organism, obtains its nutrients by absorption and reproduces by spores.
Mycology is known as study of fungal spices.
Introduction:
The following sample was drawn from an old moist wood, the sample under observation
appeared yellowish orange in color, and contained small fluffy buds. Following procedure were
followed for microscopic study of given sample.
Apparatus:

Microscope
Needles
Petri Dish
Spatula
Alcohol
Lactoglycerol
Given Fungal Sample

Procedure:
1. Take a sterilized glass slide and place a small drop of lactoglycerol in the middle of the
glass slide.
2. Prepare a petri dish with alcohol bath for needles sterilization.
3. Take the needles and sterilized them by dipping into alcohol bath, dry them.
4. Now draw out the sample from one of the needles from sample dish, and place the
sample onto the lactoglycerol drop.
5. Carefully spread the sample inside the lactoglycerol drop with the help of both needles.
6. When fully mixed into the drop, place a glass slide cover on to the sample mixture
carefully.
7. Take the prepared slide to the microscope for study.
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Observations:
The observed sample was visible at x10 magnification, the fungus identified was from the class
ascomycetes based on the microscopic structure observed.
Ascomycetes form sexual spores within a sac and are called Asco-spores. The sac is called as
ascus. They form septate hypae.
Observed specie could be Nectria cinnabarina, also known as coral spot, it thrives in dead wood
and spores are airborne. Typically occurs when there is surplus water and temperature is cold.
 EXPERIMENT NO. 14
STUDY OF FUNGI BY SIMPLE STAINING METHOD

Apparatus:
Wire loop Cover slip Given culture of Yeast
Slide box Burner
Glass slide Microscope

Chemical:

Methylene blue Safranin dye Distilled water

Theory:
Common yeast is also called baker’s yeast or true yeast. It is a unicellular fungi, yeast can be easily stained by
simple dye like safranin.
Shape of cell is oval or egg like, reproduction is mainly through binary fission or budding.

Procedure:
1. Take a sterile glass slide, and prepare smear of given yeast under sterilized conditions.
2. Add the dye onto the slide in a drop wise fashion, for at least one minute. This gives time for dye to
retain on cell.
3. Wash the slide with distilled water to remove extra dye retained on glass slide.
4. Air dry the slide and prepare to observe under microscope.

Precaution:
i. Perform the practical under sterilized conditions
ii. Sterilize all apparatus before use and after use.
iii. Air dry the slide.
iv. Be aware of dye color, and avoid touching your cloths and hands.
v. Always wear your lab coat while performing the practical.
 Structure of single Yeast Cell

Results:
Blue oval yeast cells are observed under microscope by simple staining method
with methylene blue dye.
 EXPERIMENT NO. 15
STUDY OF STERILIZATION TECHNIQUE
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