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INDEX

BACTERIOLOGY- 2073
PLP 604 3(2+1)

S. No. Name of the Practicals Date Remarks

1 Sterilization and preparation of media and buffers

2 Isolation of bacteria from diseased plant and seed


samples

3 Isolation of bacteria from soil samples

4 Preservation of bacteria

5 Observation of bacteria under microscope from


pure culture and disease samples

6 Determination of bacterial population in a colony


and bacterial suspension by optical density and
serial dilution and plating

7 Hypersensitive reaction (HR) test of bacterial


isolates on tobacco leaves

8 Pathogenicity test of bacterial isolates on host


plant (rice BLB)

9 Gram staining

10 KOH test

11 Potato soft rot test

12 Seedling symptom test of rice seeds in a cassette


holder

13 Control of seed borne bacterial diseases by seed


treatment in vitro

0
Practical No 1
STERILIZATION AND PREPARATION OF MEDIA AND BUFFERS
OBJECTIVES
To know the method of sterilization and preparation of media.
To avoid contamination during working with bacteria.
To be able to prepare media for the culture of bacteria.
To grow bacteria in vitro.

MATERIALS REQUIRED
1. Autoclave 8. Spirit lamp, spirit
2. Conical flasks (200, 500, 1000 ml) 9. Weighing balance
3. Cotton, aluminium foil, 10. Test tubes
4. Electric shaker 11. Pipette
5. Hot air oven 12. Petri plates
6. Magnet sticks 13. Heater
7. Measuring cylinder (100, 500 ml) 14. Laminar flow

Media and chemicals


1. Agar agar, yeast extract, Nutrient broth 4. NaCl, MgSO4
2. Bi-destilled water 5. Spirit (pure)
3. Ethanol (70 and 96 %)

INTRODUCTION
Media
Any substrate or substance used of microbial growth is a medium. Media may be solid, liquid
natural or artificial. However in laboratory mostly, we use artificial media. Basically there are
three types of media.
1. Standard or basic media: This media used for the culture of many types of bacteria or
fungi. It is very common media, which is not specific to any pathogen, e.g. NGA, NA,
YDC, PDA etc.
2. Semi-selective media: More or less selective to target pathogen, but may grow some
allied or related species too to some extent. It helps also to the identification of a
pathogen. More common, e.g. Cefazoline Cellobiose Methionine medium for
Xanthomonas campestris, King’s B for identification of fluorescent Pseudomonas.
Bacterium produces bluish green color in this medium facilitating easy diagnosis.
3. Selective media: Here grows only target pathogen. It is very difficult to develop. Hence,
it is not common.
Composition of some media (for a liter bidistilled water)

1
Ingredients NGA NA KB SPA YDC PDA RM TSBA
Nutrient broth 8g - - - - - - -
Glucose or dextrose 11g 2.5 g - - 20 g 20 g - -
Yeast extract 3g - - - 10 g - 3g -
CaCO3 - - - - 20 g - - -
K2HPO4 - - 1.5 g 0.5 g - - - -
MgSO4 X 7H20 - - 1.5 g 0.25 g - - - -
Glycerol (glycerine) - - 15 ml - - - - -
Peptone - 5g 20 g 5g - - 10 g -
Beef extract - 3g - - - - - -
Sucrose - - - 20 g - - - -
NaCl - - - - - - 5g -
Tryptic soy broth - - - - - - 30 g
Potato chips - - - - - 200 g1 -
Agar 20 g 20 g 20 g 20 g 20 g 20 g 20 g
NGA = nutrient glucose agar, NA = nutrient agar, KB = King et al’s B, SPA = sucrose
peptone agar, YDC = yeast extract dextrose chalk agar, PDA = potato dextrose agar, RM =
Rhodes’ medium, TSBA = tryptic soy broth agar.
1
Peel potato and cut into 12 mm cubes. Weigh 200 g, rinse in tap water, boil in 500 ml
destilled water until soft and strain through a muslin cloth. Weigh dextrose and agar and pour
in a 500 ml conical flask. Suspend in 400 ml bd-water. Measure potato extract mix in conical
flask and make a final volume of 1 litre with bi-distilled water.
PROCEDURE
 One liter water was taken in a steel pan and placed okn heater for boiling.
 20g of dextrose or glucose was added and stirred
 10 g of Yeast extract was added and stirred again
 20g of CaCO3 (not precipited0 was added in that pan and stirred continuously.
 20g Agar was added and stirred continuously until the agar melts. Agar melts at 100 0 C
when drop medium was placed in cement floor if agar melts then it coagulate otherwise
not.
 Sterilize the tube was taken and poured the medium in that test tube so that each content
5ml. pouring was done in laminar floor which as sterilized with alcohol or spirit.
 Test tube mouth was plug with sterilized cotton.
 This test tube mouth was covered with aluminium foil and put in autoclave for
sterilization. It was put in autoclave for 15 minutes at 1210C.
 After then, those test tubes were placed in slanting position.
Buffers: For bacteriological work, we use buffer. It helps to maintain the situation constant.
It helps to protect the bacterial cells from osmosis, i.e. plasmolysis. 0.85 % NaCl or 0.01

2
Molar MgSO4 (i.e. 2.46g/l) require to maintain osmotic pressure of bacteria, i.e. to check up
plasmolysis of the cells.
Sterilization
Sterilization means complete destruction of all living organisms by heat, with or without
pressure, chemicals, filtration, irradiation and ultrasonic vibration. It is done to make
something free from pathogens or microbes. There are following methods of sterilization.
1. Dry air: glass wares such as Pertriplates, testtubes, or flasks or pipettes are sterilized
by heating at about 160-170C for 1-4 hours in a hot air oven. Thjermostat helps in
regulating temperature in oven.
2. Stem under pressure (Autoclaving): usually the media in test tubes or flasks are
autoclaved at 10lb pressure at 115C for 30 minutes, 15lb pressure (121C) for 15
minutes or 20lb pressure at 126C for 10 minutes depending upon the amount on
media present.
3. Filtration: All type of media can’t be sterilized by autoclaving and hot air methods.
Certain sugar, amino acids and vitamins are heat liable materials and this methods in
commonly used in the phytobacteriology. Various types of filter are Seitz filter,
Chamber land filter, Mender filter, ultra filter, membrane etc. all filters have the
sufficient pores to trap and remove the microorganisms from liquid media.
4. Chemifals: chemicals like dettol, phenol, mercuric chloride (0.01%) ethyl alcohol
(75%) potassium dichromate, sodium hypochloride are antimicrobial agents and are
often used for surface sterilizations. The sterilization material is dipped for 1-2
minutes in chemicals and then washed off with the sterilized distilled water.
5. Flaming: Pipettes, needle, scalpel scissors forceps and other metallic instruments are
dipped in ethanol or spirit and burned in flame.
6. Others: the gamma and ultraviolet (UV) rays can kill the microorganisms. The
isolation chambers can be sterilized by putting the UV rays on for a few minutes.
Certain plant tissue can be sterilized by fumigating with propylene oxide.

Cefazoline Cellobiose Methionine Medium (CCMM) (a semi selective medium for Xcv)
Ingredients Quantity
1. Cellobiose 10 g
2. NH4Cl 1g
3. KH2PO4 0.4 g
4. K2HPO4 1.5 g
5. MgSO4 X 7H2O 0.3 g
6. D-L Methionine 1g
7 Cefazoline 10 mg
8. Cycloheximide 200 mg
9. Agar 14 g
10. Bd water 1000 ml

3
Composition of Crystal violet (poly) pectate (CVP) medium
Ingredients Quantity
1. Crystal violet solution (0.075 %) 1 ml
2. NaOH (1 N = 8 g NaOH/200 ml dist. Water) 4.5 ml
3. CaCl2 X 2H20 (10 %) 6 ml
4. NaNO3 1g
5. Trisodium citrate dihydrate 2.5 g
6. Sodium polypectate 9g
7. Sodium lauryl sulfate (10 %) 0.5 %
8. Agar 2g
9. Distilled water 500 ml
Receipe of D-1 agar (Agrobacterium grows but not Pseudomonas on this medium)
Ingredients Quantity
1. Mannitol ` 15 g
2. NaNO3 5g
3. LiCl 6g
4. Ca(NO3)2 X 4H2O 0.002g
5. K2HPO4 2g
6. MgSO4 X H2O 0.2 g
7. Bromthymol blue 0.1 g
8. Agar 15 g
9. Distilled water 1000 ml

CONCLUSION
Thus, we became able to prepare different types of media for preservation of cultures of plant
pathogens (bacteria and fungi) and

4
Practical No. 2
ISOLATION OF BACTERIA FROM DISEASED PLANT AND SEED SAMPLES

OBJECTIVES
1. To diagnose a bacterial disease.
2. To identify an ideal method of bacterial isolation.
MATERIALS REQUIRED
1. Bent glass rod 9. Parafilm
2. Blade, knife, scissor, forcep 10. Pestle and mortar
3. Conical flasks (200, 500, 1000 ml) 11. Petriplate rotator
4. Electric shaker 12. Spirit lamp
5. Glass tubes (with 4.5 ml buffer) 13. Weighing balance
6. Incubator (29 C) 14. Wire loop
7. Measuring cylinder (100, 500 ml) 15. Centrifuge
8. Micropipettes (100 l, 500 l), pipette 16. Electric shaker
tips 17. Forceps
Seeds and plant samples
Media and chemicals
1. Bi-distilled water 2. Ethanol (70 and 96 %)
3. Buffer: NaCl (0.85 %) or MgSO4 (0.01 M) 4. Media: NGA, YDC, KB etc
5. Spirit (pure)

INTRODUCTION
Bacteria are commonly carried on seed, and potentially may be seed transmitted. In the most
of the annual crops which is attacked by PPB are survived on the seed and normally seed
transmitted. The seed borne bacteria can survive in the seed and serves as the source of
inoculum and may cause bacterial disease epidemics. The bacteria are often carried on the
surface of the seed but bacteria causing vascular or systemic infections are frequently found
in seed coat or other part of the seed. For instance, Pseudomonas phaseolicola and X.
phaseoli in Phaseolus bean are found in the hilum region of the seed into which they
penetrate from the vascular system through the funiculus. Bacteria borne on the surface of the
seed mauy keep alive for a limited period of the time only, perhaps one to two years, where as
bacteria harboured within seed tissues may live many years for ex. Corynebacterium
flaccumfaciens which has survived 24 years in phaseolus bean seed kept under lab condition.
Most commonly known seed borne bacterial genus are: Pseudomonas, Xanthomonas,
Erwinia and Corynebacterium.

5
Methods for isolation of bacteria from seeds:
1. By soaking seeds overnight (12hr) in buffer solution at 250C under stirring
In dry seeds bacteria are inactive. When they go they get enough moisture and proper
temperature, they become active and start to move in water. They may also release some
substance and they become identified.

2. By grinding and serial dilution


When the seeds are crushed, the tissue of the seed will be damaged and bacteria will
automatically release from the seed themselves or by force. Generally if we need the quick
extraction of the bacteria, grinding is practiced. Small seeds are generally grinded and large
seeds are soaked such as the seed of soybean, bean.
Methods of isolation from plant samples
1. Serial dilution
2. By direct streaking with diseased sample
3. By streaking ooze
PROCEDURE
1. Grinding method
 Take seed sample (1 gm)
 Grind the seed sample in the mortar and pestle in buffer solution (1 ml). The seed
sample should be finely crushed
 Make three solutions (10-1, 10-2, and 10-3) from the crushed slurry.
 Take 0.5 ml suspension from each dilution and plate it in the NGA plate.
 Incubate the plate at 250C and observe the colony after 24-48 hrs.
 Record the colony characters their number and isolates the pure colony for the further
diagnosis.
2. Soaking method
 Take seed sample and weigh it
 Soak the seed overnight and centrifuge it at 50000 rpm for 10 minutes.
 Remove the supernatant and take the remaining suspension.
 Make the three dilutions (10-1, 10-1 and 10-3 from the suspension.
 Take 0.5 ml suspension from each dilution and plate it in the NGA plate.
 Incubate the plate at 25 0C and observe the colony after 24 – 48 hrs.
 Record the colony characteristics, their number and isolate the pure culture colony for the
further diagnosis.

6
OBSERVATION
Samples were citrus canker from leaves and stem, rice leaves, rice seeds
(old and new).
SN Sample Colony character
1 Rice old seed Yellow, slimy
2 Rice new seed Whitish
3 Rice leaf Whitish
4 Citrus leaf fresh Yellow
5 Citrus leaf (dry) No bacterial growth
6 Citrus stem No bacterial growth

CONCLUSION
Hence we isolated bacteria from different plant and seed samples.

7
Practical No. 3
ISOLATION OF BACTERIA FROM SOIL SAMPLES
OBJECTIVES
1. To determine whether the soil is infected with plant pathogenic bacteria.
2. To identify prevailing plant pathogenic bacteria.
3. To find out types of bacteria occurring in the soil.

MATERIALS REQUIRED
1. Bent glass rod 7. Permanent marker
2. Electric shaker 8. Petriplate rotator
3. Glass tubes (with 4.5 ml buffer) 9. Soil sample (1 g)
4. Incubator (29 C) 10. Spirit lamp
5. Measuring cylinder (100, 500 ml) 11. Weighing balance
6. Micropipettes (100 l, 500 l), pipette tips 12. Wire loop

Media and chemicals


1. Bi-destilled water 4. Media: NGA, YDC, KB etc
2. Buffer: NaCl (0.85 %) or MgSO4 (0.01 M) 5. Spirit (pure)
3. Ethanol (70 and 96 %)

INTRODUCTION
Many saprophytic and plant pathogenic bacteria will survive in the soil. Plant pathogenic
bacteria like Xanthomonas, Pseudomonas etc can live in soil. Thus isolation of PPB from soil
gives some idea to estimate the soil; inhabiting bacterial population in the soil.
As most of the bacteria being the facultative saprophytic in nature, they don’t survive in soil
for the longer period but some bacteria like Ralstonia, Streptomyces etc survive in soil. They
mainly present in the soil in plant debris and even in oozing drop for the short period,
Antagonistic bacteria are also found in soil.
Thus the semi selective media can be used to grow the pathogen. They survive at 15-30 cm
depth and under favorable soil condition i.e. aeration, moisture, porosity etc, it may found
rather deeper layers.

PROCEDURE
Bacteria is isolated by serial dilution of soil samples:
 Collect the soil samples form different site of he fields (both rhizosphere and non
rhizosphere soil). The soil sample should be taken below the 15 cm soil depth.

8
0.5 ml 0.5 ml
0.5 gm soil

4.5 ml 4.5ml 4.5 ml 4.5ml 4.5ml 4.5ml

10-1 10-2 10-3 10-4 10-5 10-6

Fig. Serial dilution of soil

Spreading rod

NGA medium

Petriplate

Fig: Spreading of bacterial suspension NGA medium

9
Prepare the composite sample by mixing the collected samples from the different sites and
take 0.5 gm soil from it.
 Label the dilution blanks as 10-1, 10-2, 10-3, 10-4, 10-5, 10-6 and 10-7.
 Prepare the initial dilution by adding 0.5 ml or 0.5 gm of the sample (1 colony) into 4.5
ml dilution blank labeled as 10-1 thus diluting the original sample 10 times.
 Mix the contents by vertexing the tube on the vertex equipment for uniform distribution
of the pathogen on the suspension.
 From the first dilution, transfer the 0.5 ml suspension while in motion, to the dilution 10 -2
with a steriloe and fresh micropipette (0.5 ml) diluting the original specimen/ suspension
to 100 times (10-2).
 Repeat the procedure till the original sample has been diluted 1000000(10-6) times using
every time a fresh sterile pipette tubes.
 From the appropriate dilutions (10-1 to 10-7) transfer 1 ml or 0.1 ml of the suspension
while in motion, with the respective pipettes, to sterile petridishes containing NGA. Three
petridishes are to be used foe each dilution (if 0.1 ml is plated; the dilution is increased 10
times).
 Incubate these plates in an inverted position for 24-48 hrs at 37 0C.
 Observe the colony after 24-48 hrs incubation.

RESULTS
Soil sample was taken from plant pathology field and diluted (10 -1 to 10-6). They (100µl) were
transferred to NGA plates and spreaded on it. The colonies were observed after incubation for
48 hrs. The characteristics of the colony and their number were recorded.
Table Bacterial colonies observed on the soil sample

Sample No Colony character No. of colonies in


10-5 10-6
1 Whitish 130 26
2 Yellowish 213 102

CONCLUSION
Thus from this practical we isolated bacteria from soil samples.

10
Practical No. 4
PRESERVATION OF BACTERIA

OBJECTIVES
1. To know the method of preservation of plant pathogenic bacteria
2. To be able to preserve plant pathogen for further use and test

MATERIALS
1. Electric shaker 4. Parafilm
2. Incubator (29 C) 5. Spirit lamp
3. Measuring cylinder (100 ml) 6. Wire loop
7. Glass tubes (with 10 ml buffer or bidest. water)
Media and chemicals
1. Bacteria: pure cultures 4. Ethanol (70 and 96 %)
2. Buffer: NaCl (0.85 %) or MgSO4 (0.01 M) 5. Media: NGA, YDC slant agar etc
3. Bi-destilled water 6. Spirit (pure)

INTRODUCTION
Different methods of preservations are:
1. In Petri plates
Seal in the paraffin and store about 5 oC.

2. Dry agar plates


The cultures are grown in test tubes or bottles on a nutrient medium, usually an agar gel with
added nutrient. The nutrients may be pieces of vegetable or plant material, vegetable
decoctions, or mineral salts plus carbohydrate source. One of the most useful is a very weak
decoction of potato carrot extract and agar. These simple dry cultures, the standard laboratory
cultures, may be stored.
When bacteria utilize the nutrient makes the media acidic and 16 hour use of the CaCO 3 in
YDC maintain the change on PH of media.

3. At room temperature
This best done in a wooden cupboard to protect the culture from dust. They may dry out
particularly rapidly in tropical climates and must be transformed to fresh media at least every
6 months. In a refrigerator or cold room at 5-8 0C. again it is necessary to transfer at least
every 6 months. Some strains are sensitive to cold. In a deep freeze at about -20 0C. when
placed in a deep freeze survival is for a longer period and has proved very satisfactory.

11
4. Distilled and autoclaved water storage
A double pollination of bacteria can be preserved for about 1 year at 10-20 0C room
temperature. From this method Ralstonia can be easily stored. There has been a recent
interest in possible alternative cheap and simple methods of storage. In one Boewinkel and
Ellis after Castellani store squares, cut from the edge of young fungal cultures on agar, in
sterile distilled water in screw cap bottles. The caps are immediately screwed down and the
bottles stored at room temperature. Good revivals for several years has been obtained for
many fungi and it has been found at the CMI particularly useful for Phytophthora and
Pythium species.

5. Freeze drying and lyophilization

Glass tubes air


tight or glass
ampoule

Bacteria
Silica gel
Fig: Lyophilized bacterial culture

Cultures are dried from the frozen state by with removing the water vapour under reduced
pressure. The dried cultures or spore suspensions are sealed and stored in glass ampoules.
This is best studied to healthy sporing cultures; mycelial forms Phytophthora and Pythium
species don’t survive the initial treatment. The final cultures are completely sealed in glass,
so there is no risk of cross infection or mites. The apparatus involved tends to be costly and
complex. If the fungi survive the initial treatment, they should be viable for 10 more years.

Liquid nitrogen
cultures, tissue or spore suspension are treated with the protective medium as 10% glycerol,
place in din ampoules and frozen at ultra low temperatures such as in liquid nitrogen.
Revivals are said to be the best when the cooling is done slowly. At these low temperatures
metabolism is suppressed, so if the initial shock of freezing is survived the cultures should
remain viable indefinitely. This technique requires large and expensive apparatus as well as
reliable source of liquid nitrogen.

CONCLUSION
Thus from this practical, we became familiar with the different methods of preservation of the
culture media for fungi and bacteria.

12
Practical No. 5
OBSERVATION OF BACTERIA UNDER MICROSCOPE FROM PURE CULTURE
AND DISEASE SAMPLES
OBJECTIVES
1. To identify a bacterial cell.
2. To diagnose whether a disease was caused by bacteria.

MATERIALS REQUIRED
1. Blade, knife, scissor, forceps, needle 4. Slides and cover slips
2. Cotton 15. Spirit lamp
3. Microscope 16. Wire loop
Media and chemicals
1. Bacteria: pure cultures 4. Ethanol (70 and 96 %)
2. Buffer: NaCl (0.85 %) or MgSO4 (0.01 M) 5. Spirit (pure)
3. Diseased plant parts
Sources of bacteria
1. Pure cultures
2. Diseased plant tissues
INTRODUCTION
Plant pathogenic bacteria can be seen under the microscope by preparing the semi permanent
slide. When a plant is infected with the PPB, we can observe the pathogen from the symptom
developed area. It will help to know the probable pathogen on the infected plants. Bacterial
population should be counted for the determination of the bacterial population in the prepared
culture or on the colony of the bacteria.

PROCEDURE
 Take a bacterial cell from the colony with the help of loop and keep it on the clean slide
containing a drop of water.
 Cover the cell suspension with the help of neat cover slip.
 Observe it under the compound microscope at different magnification (10 xs, 40 xs, and
100 xs).

OBSERVATIONS
Bacteria were also observed under the compound microscope. The bacterial cells from colony
were taken and slide was prepared and bacteria were observed in 10x, 40x, and 100x
magnification. The moving bacterial cell was seen under 40x and 100x magnification in
compound microscope.

CONCLUSION
Thus we observed bacteria under compound microscope.

13
Practical No. 6
DETERMINATION OF BACTERIAL POPULATION IN A COLONY OF
BACTERIAL SUSPENSION
OBJECTIVES
1. To determine whether all types of colonies contain similar number of bacterial cells.
2. To find out an easier and reliable method of determination of bacterial population in a
suspension or colony.

MATERIALS REQUIRED
1. Bent glass rod 9. Paper towel
2. Colony counting chamber 10. Permanent marker
3. Dropper 11. Petriplate rotator
4. Electric shaker 12. Spectrophotometer
5. Glass tubes (with 4.5 ml buffer) 13. Spirit lamp
6. Haemocytometer 14. Wire loop
7. Incubator (29 C) 15. Micropipettes (100 l, 500 l), pipette tips
8. Microscope

MEDIA AND CHEMICALS


1. Bacteria: pure cultures
2. Buffer: NaCl (0.85 %) or MgSO4 (0.01 M) in test tubes
3. Ethanol (70 and 96 %) or Spirit (pure)
4. Media: NGA, YDC etc

METHODS
1. Serial dilution agar plate technique
The plate count technique is one of the most routinely used procedures because of the
enumeration of viable cells by this method. This method is based on the principle that when
material containing bacteria are cultured, every viable bacterium develops into a visible
colony on a nutrient agar medium. The numbers of colonies, therefore, are the same as the
number of organisms contained in the sample. In this procedure, a small measured volume or
weight is mixed with a large volume of sterile water or saline called the diluent or dilution
blank. Dilutions are usually made in multiples of ten. A single dilution is calculated as
follows:
Dilution = volume of the sample
Total volume of the sample and the diluent

14
Serial dilutions are later prepared by transferring a known volume of the dilution to second
dilution blank and so on. Once diluted, the specified volume of the dilution sample (1 ml
or 0.5 ml) from various dilutions is added to sterile plates (in triplicate for each dilution)
to which molten and cooled (45-500C) suitable agar medium is added. The colonies are
counted on a Quebec colony counter. The number of organisms developed on the plates
after an incubation period of 24-48hrs/ml is obtained by multiplying the number of
colonies obtained per plate by the dilution factor, which is reciprocal of the dilution. To
facilitate calculations, the dilution is written in exponential notation. For example 1:1000
dilutions would be written as 10-3.
Number of cells/ml = Number of colonies
Amount plated x dilution
2. Using spectrophotometer
Bacterial population or amount of growth can be determined by measuring turbidity or
optical density (i.e. cloudiness of the suspension) of a broth culture. Since turbidity is
directly proportional to the number of cells, this property used as an indicator of bacterial
concentration in sample. Turbidity is quantified with the spectrophotometer that
measures the amount of light transmitted or absorbed directly through a sample. The
density of the cell suspension is expressed as absorbance or optical density (a value
derived from the percentage or transmission) which is directly proportional to the cell
concentration. Absorbance is a logarithmic value and used to plot bacterial growth on a
graph. Generally, at 660nm, if 0.06 optical density of the concentration then the
population will be 108 cfu/ml.
Absorbance = -log % Turbidity
100
3. Using Haemocytometer
Haemocytometer is used to count the fungal spore or bacterial cells in liquid suspension. It is
a special microscope slide with a counting chamber 0.1 mm deep so that volume of liquid
over a one sq. mm is o.1 cubic mm. the counting chamber has total of nine squares, each of
1mm x 1mm engraved over it. But only one square per field is visible under 100 x
microscope magnifications (10 x ocular and 10 x objectives). One mm square is divided in to
25 medium sized squares, each of which is further subdivided in to 16 small squares, thus a
total of 400 squares in 1mm. triple lines separate each medium – sized square, the middle
one act as the boundary. Each large square has a volume of 1 x 1 x 0.1 mm (1/10 x1/10
x1/100 cm) = 1/10000 cm3 or 10-4 cm3. One ml of a cell suspension is put in to the counting,
chamber, numbers of cells is counted and the total cell number is determined mathematically.

Serial dilution and plating on a media


 Pure culture of bacterial colony was taken
 5 ml of MGSO4 was taken in a test tube and a bacterial colony was mixed with
distilled water by smoothly rubbing on test tubes wall by means of inoculating needle
 0.5 ml of buffer solution was taken and 0.5ml of suspension from the first tube was
put in to the test tube, serial dilution was maintained upto 10-6
 Optical density of diluted bacterial suspension was measured
 When optical density is 0.06 at 660 nm then bacterial suspension is 1 x 108 cfu/ml
 To maintain OD Pearson square method is used

15
We have 0.2 OD but we need 0.006 OD, which can be calculated as follows

Bacterial 0.06ml x
Suspension 0.2 100 = 6
ml

0.06

Water 0 0.14 x 100


= 14ml

-in 0.06 OD, bacterial concentration is 108.


-we need about 150-200 bacterial colony otherwise they join to each other.
-If we require 10-5 bacterial suspension to maintain this amount of colony.
- For this we have to dilute 10-5 to 10-6.
-10-5 dilution of 50 ml 140 cfu.
-To maintain 280 cfu 100ml of 10-5 dilution should be taken.
-To observe the bacterial colony plating should be done.

CONCLUSION
Thus, we became able to determine the bacterial cell in a colony and in a suspension.

16
Practical No. 7
HYPERSENSITIVE REACTION (HR) TEST OF BACTERIA ON
TOBACCO LEAVES
OBJECTIVES
1. To determine whether the bacteria/isolates are pathogenic or not.
2. To identify possible bacteria.

MATERIALS
1. Electric shaker 5. Spectrophotometer
2. Micropipettes (100 l, 500 l), pipette tips 6. Spirit lamp
3. Paper towel 7. Syringe and needles
4. Permanent marker 8. Wire loop
Media and chemicals: Bacterial pure cultures
Buffer: NaCl (0.85 %) or MgSO4 (0.01 M) in test tubes
Ethanol (70 and 96 %) or Spirit (pure)
Plants: Tobacco
INTRODUCTION
Hypersensitive reaction is a localized induced cell in the host plant at the site of infection by
a pathogen. It is thought to be responsible for limiting the growth of pathogen and, in that
way, is capable of providing resistance to the host plant against the pathogen. Injection of
several genera of plant pathogenic bacteria into leaf tissue of non host plant result in the
development of hypersensitive response. HR consists of a large leaf sector becoming water
soaked at first and, subsequently, necrotic and collapsed within 8-12 hrs after inoculation.
The bacteria injected in the tissue are trapped in the necrotic lesion and generally are killed
rapidly. The HR may occur whenever virulent strains of plant pathogenic bacteria are injected
into non host plant or into resistant varieties, and when avirulent strains are injected into
susceptible cultivar.
PROCEDURE
We have taken different isolates of bacteria (taken from seed and plant sample of rice). Then
we made suspension of 5 x 10 6 cfu/ml of bacterial cell by using spectrophotometer at 0.06
optical densities at 660 nm. Then, we injected those bacterial suspensions on tobacco leaves.
Tag was given individually and observed after 1 day.
OBSERVATION
Tobacco plant (leaves) showed no Hypersensitivity Reaction.
CONCLUSION
Hence, we perform the hypersensitivity reaction of bacteria on tobacco, but all bacteria
showed HR negative.

17
Vein

Syringe

Fig. Inoculation of bacterial suspension on leaf

18
Vein

Hypersensitive
reaction

Fig. Hypersensitive reaction on tobacco leaf

Practical No. 9
GRAM STAINING

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OBJECTIVES
1. To know the procedure for preparation of staining solution.
2. To determine whether the new bacteria are Gram positive or negative
3. To compare Gram stain with KOH test for efficiency.

MATERIALS
1. Conical flasks (100, 500 ml) 7. Permanent marker
2. Cotton 8. Pestle and mortar
3. Dropper 9. Slides
4. Magnet sticks 10. Spirit lamp
5. Magnet stirrer 11. Weighing balance
6. Measuring cylinders (100, 500 ml) 12. Wire loop
Media and chemicals
1. Bacteria: pure cultures 3. Immersion oil
2. Distilled water 4. Methanol

SOLUTIONS FOR GRAM STAINING


1. Staining solution (Hucker's ammonium oxalate crystal violet)
1.1. Solution A
Crystal violet (Difco, > 90 % dye content) 2.0 g
Ethanol (ethyl alcohol: 95-96 %) 20.0 ml
1.2. Solution B
Ammonium oxalate (Fisher) 0.8 g
Distilled water 80.0 ml
Mix solutions A and B and filter through a filter paper.

2. Fixing solution (use fresh)


NaHCO3 (optional, may be excluded) 1.5 g
Iodine 0.5 g
Potassium iodide 1.0 g
Distilled water 150.0 ml
Grind first all 3 ingredients together in a pestle and mortar and then dissolve in distilled.
water. Dissolution may take more than 1 h.
3. Counter-staining solution

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3.1. Stock solution
Safranin (Fisher) 2.5 g
Ethanol (95-96 %) 100.0 ml
3.2. Working solution
Stock solution 10.0 ml
Distilled water 90.0 ml
Important points to be considered in successful staining:
1. Use reagents < 1 year old. Especially, iodine solution should be put in a brown bottle or
kept in dark or wrap with aluminum foil. Otherwise, it will be degraded and decolorized
due to light.
2. Use bacteria of exponential growth phase. Cells of stationary phase may give a Gram
variable reaction.
3. Use known Gram positive and Gram negative bacteria as a control, and
4. Bacterial smear must show some well separated groups of bacteria. Bigger clumps hamper
proper staining.
PROCEDURE
1. On a clean slide (fat free), dry a thinly spread bacterial smear form colony or thick
suspension in air. Lightly flame the underside of the slide twice to fix the bacteria on the
slide. Bacteria may also be fixed by dipping slides a few seconds in methanol and by air
drying. Optimum fixing kills and attaches bacteria to the slide, but will not distort the
structure.
2. Flood the smear with staining solution, i.e. crystal violet and let to stain for 1 min.
Bacterial cells carry a slightly negative charge, so that they combine best with dyes carrying
a positive charge, such as, crystal violet, methylene blue, carbol fuchsin and safranin. Of
these, crystal violet is the best, because it takes very little time for penetration.
3. Wash in tap water or with fixing solution few seconds. Drain off excess water, and lightly
air dry.
4. Flood the smear with fixing (iodine) solution for 1 min. It fixes crystal violet dye in
bacterial cells.
5. Wash in tap water a few seconds and blot dry.
6. Destain with ethanol (95-96 %) repeatedly until the solvent (ethanol) flows colourlessly
from the slide (10-30 secs). Blot dry. If decolourizer is used longer, even Gram positive
bacteria may lose colour.
7. Rinse in tap water for about 5 secs.
8. Counter-stain with safranin working solution for ca. 10 to 60 secs.
9. Wash briefly in tap water, air dry and examine under oil immersion (100 x) objective.
Gram positive bacteria appear blue and Gram negative bacteria red.

Practical No. 10

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KOH (POTASSIUM HYDROXIDE) TEST
OBJECTIVES
 To be familiar with the procedure of KOH test
 To compare Gram stain with KOH test for efficiency.

MATERIALS
 Slides
 Wire loop
 Bacterial colony
Chemicals: Potassium hydroxide (KOH) solution (3%)

INTRODUCTION
It is a faster and easier method used instead of Gram staining to identify Gram positive and
Gram negative bacteria. With a sterile loop fresh bacteria are mixed with 1-2 drops of KOH
solution (3 %) on a slide. When the viscocity of the suspension increases (due to cytoplasmic
fluid) very highly within a few seconds, and slime threads are formed during pulling up of the
loop from the suspension, the bacteria are Gram negative. If the viscocity does not or very
weakly increases, the bacteria are Gram positive.
Due to thinner cell wall and different composition, Gram negative bacterial cells are
dissolved in KOH-solution and DNA might also be playing a great role in the formation
of slime threads, or increment of viscosity.

PROCEDURE
With a sterile loop fresh bacteria were mixed with 1-2 drops of KOH solution (3%) on a
slide. After mixing, the loop was pulled from the slide. And observed the viscosity and
thread formation.

OBSERVATION
Bacteria isolated from leaves and stem, rice leaves, rice seeds (old and new) were used for
KOH test. All the tested bacteria showed KOH negative results.

CONCLUSION
By this practical, we are acquainted with KOH test.

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Practical No. 11
POTATO SOFT ROT TEST

OBJECTIVES
 To know about the potato soft rot test.
 To identify the bacteria.

MATERIALS REQUIRED
Ethanol, potato tubers, knife, filter paper, petridishes, inoculation loop, incubator etc.

INTRODUCTION
Certain bacteria such as P. marginalis, E. carotovora group (E. carotovora sub sp.
carotovora, E. carotovora sub sp. artoseptica), E. chrysanthemi produces pectinase enzyme
and degrades middle lamella (composed of pectin) resulting in soft rot especially in potato
tubers or disks. Some strains of Pseudomonas, Xanthomonas, Bacillus and Clostridium also
cause weak soft rotting on potato slices.

PROCEDURE
1. Select fresh, nonsprouting, healthy potato tubers and wash well,
2. Remove rind and cut about 8 mm thick slices,
3. Surface sterilize flaming with ethanol,
4. Place a water soaked, sterile filter pater in a Petridish,
5. Place slices in Petridish separately,
6. Inoculate the slices at the centre with a pure culture of bacteria with a loop,
7. Do not forget control slice one in each plate,
8. Incubate at ca. 20 to 28 °C for 24 to 48 h,
9. Test rotting with a sterile loop. When the inoculated portion is soft remaining the
surrounding portion hard, the test is positive, if all the parts are hard the test is negative,
10. Smell whether the rotten slices stink. Foul smell is due to secondary (saprophytic) attack,

OBSERVATION
Bacteria isolated form old rice seed (1) new rice seed (2), rice leaf (3) and citrus fresh leaf (4)
was used for HR test.
Table Observation of potato soft rot test
Sample No Sample Effect of pathogen (soft rot)
1 Old rice seed
2 New rice seed
3 Rice leaf
4 Citrus fresh leaf

CONCLUSION
Thus we conducted potato soft rot test.

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Practical No. 12
ISOLATION OF ANTAGONISTYIC BACTERIA FROM DISEASED PLANT AND
SOIL AND TEST OF ANTAGONISM IN VITRO
OBJECTIVES
1. To determine whether the soil has any antagonistic bacterium.
2. To compare morphology of antagonistic and plant pathogenic bacteria.
3. To compare antagonistic bacteria isolated from plant and soil samples.
4. To compare effectiveness of antagonistic bacteria against various types of
pathogenic or nonpathogenic isolates or bacteria.

MATERIALS
1. Blades, knives, forceps 6. Permanent marker
2. Bent glass rod 7. Pestle and mortar
3. Electric shaker 8. Spirit lamp
4. Micropipettes (50 and 500 l) 9. Weighing balance
5. Needles 10. Wire loop
11. Diseased plants and soil sample

Media and chemicals


1. Buffers in test tubes 3. Spirit or ethanol
2. NGA plates

INTRODUCTION
Antagonism is a phenomenon of interfering or killing of one organism by another organism
and the pathogen which kills another pathogen is called as antagonistic organism. Many
bacterial pathogens are antagonistic to other bacteria. Mostly saprophytic bacteria or avirulent
strain of pathogenic bacteria are antagonistic to PPB. For e.g. A. tumefaciens can be
controlled commercially by treating the seeds, seedlings and cuttings, a suspension of strain
K-84 of related but non-pathogenic A. radiobacter. Similarly Bacillus subtulis strain A-13 for
Streptomyces are antagonistic to root pathogen. Fire blight of apple blossom caused by E.
amylovora is partially controlled with E. herbicola, bacterial leaf streak of rice caused by X.
campestris pv. oryzicola, was reduced with spray of isolates of Erwinia and Pseudomonas.

PROCEDURE
1. Isolate and purify as many types of bacteria (pathogenic or nonpathogenic) as possible
from plant and soil samples. Observe, whether some colonies appear antagonistic. Usually,
whitish, reddish, flat, wrinkled etc colonies may be antagonistic.

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2. Inoculate NGA plate at the centre with a single line of the possible antagonistic
bacterium. Conduct each test on separate plates. All other pathogenic bacteria streak on a
single line on one or both sides ca. 0.5 – 1 cm away from the line of antagonistic
bacterium by making perpendicular lines on the antagonistic bacterium.
3. Inoculate and observe whether growth of any bacterium is suppressed. If yes, then test
further against other pathogenic bacteria and fungi as well as by other methods.

RESULTS
Anta- Repli- Effects on pathogen Remarks
c
gonist 1 2 3 4 5 6
a
ti
o
n
A1 R1 - - - - - - No effects
R2 - - - - - - ,,
R3 - - - - - - ,,
A2 R1 - - - - - ,,
R2 - - - - - - One saprophytic colony suppressed two bacteria.
R3 - - - - - - No effects
A3 R1 - - - - - - ’’
R2 - - - - - - Inoculated at improper distance (long distance)
R3 - - - - - - No effect

CONCLUSION
Here, the tested bacteria were not antagonistic to the plant pathogenic bacteria.

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Practical No. 13
CONTROL OF SEED BORNE BACTERIAL DISEASES BY SEED TREATMENT
OBJECTIVES
 To detect and destroy bacterial inocula from seeds.
 To develop appropriate control mechanism for the control of seed borne bacteria.

MATERIALS
1. Bent glass rod 5. Pestle and mortar
2. Conical flasks (100, 200, 500 ml) 6. Spirit lamp
3. Micropipettes (50 and 500 l) 7. Water-bath
4. Muslin clothes 8. Weighing balance

Media and chemicals


1. Bacteria: pure cultures, to treat seeds.
2. Buffers in test tubes
3. Destilled water
4. NGA plates
5. Spirit or ethanol

INTRODUCTION
Most of bacteria are facultative saprophytes. They are mostly soil borne (soil inhabitant and
invaders), debris borne and seed borne. On plants they live epiphytotically in buds, wounds,
exudates and leaf surfaces. Seeds are the important sources of bacterial transmission. For the
bacteria which are seed inhabitant, can be controlled by the treatment of seeds. Common
methods which can be used for the control of bacteria through seed treatment as follows:
Bacteria are commonly carried on seed, and potentially; may probably may seed transmitted.
In the most of the annual crop which is attacked by PPB bacteria are survived on the seed and
serve as the source of pathogen inoculums and may cause the bacterial disease epidemics.
The bacteria are often carried on the surface of the seed but bacteria causing the vascular or
systemic infections are frequently found in seed coat or other part of the seed. For instance,
Pseudomonas phaseolicola and Xanthomonas phaseoli in the phaseolus bean are found in the
hilum region of the seed, into which they penetrate from the vascular system through the
funiculus. Bacteria borne on the surface of the seed may keep alive for a limited period of the
time only, perhaps one or two years, where as bacteria harboured within seed tissues may live
many years for eg. Corynebacterium flaccumfaciens which has survived 24 years in
Phaseolus been seed kept under laboratory conditions. Most commonly known seed borne
bacterial genus are; Pseudomonaa, Xanthomona, Agrobacterium, Eriwinia, and
Corynebacterium. Seed treatment help to control of seed borne bacterial diseases up to some
extent.

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Different methods of seed treatment arte as follows:
1. Hot water treatment (at 50 C for 30 min, or 52 C, for 20 min)
2. Solar heat treatment (soak seeds overnight in tap water and dry under hot sun next whole
day or more.
3. Chemical treatment. Seed treating chemicals are as below:
i. Sodium hypochlorite: 0.5 – 1 % for 30 min seed dipping against external infection.
ii. HCl: 0.6 Molar, for 1 h. common against Clavibacter spp. of tomato, potato.
iii. Boric acid: 3 % for 30 min against potato tuber bacterial diseases, e.g R.
solanacearum, Erwinia spp. etc.
iv. Cupric acetate:
v. Antibiotics: Streptomycin(100-200 ppm)

PROCEDURE
1. Confirm natural seed infection by isolation. If the seeds are not infected, then inoculate
seeds artificially by dipping seeds in a bacterial suspension of 108 cfu/ml for at least 1 h.
2. Treat the infected seeds with hot water by packing seeds in a muslin cloth, by solar heat
treatment and chemical method.
3. Isolate bacteria again from all treated seeds to confirm the effectiveness of the treatments.

CONCLUSION
Hence, we became acquainted with different methods of seed treatment to control seed borne
bacterial diseases.

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