Identification of bacteria
After isolation of bacteria in pure culture from a specimen it has to be identification.
Bacteria are identified routinely by morphological and biochemical test supplemented as need by specialised test such as Syria type antibiotic.
.jpeg "identification of bacteria")
Morphology of bacteria Colony the following features of the calling our study
- Size
- surface
- shape
- colour
- haemolysis.
Growth in liquid media
Media bacteria growth may be a following forms
- Uniform terbidity
- Deposits of bottom
- Surface pelical formation
Stanning of Bacteria
Staining method are emploed to examine. Smell prepared from the bacteria Colony or liquid culture commonly used as follows.
- Gram stain
- Albert stain
- Zn staining
Heinging drop preparation
Differentiate motile bacteria from non motile onces
Bacteria test
The widely used biochemical test are described below
Sugar fermentation
It determines the ability of an organism to permit a specific carbohydrate in corporated in a medium producing acid acid with gas
Observation
- Positive pinkish red
- Negative yellow to colourless
Carbohydrate fermentation test
Carbohydrate fermentation test
List of biochemical test
- Carbohydrate fermentation test
- sugar mannitol fermentation test
- Catalyst test
- IMVIC test
- Huge and Leifson's test
- Indole producing test
- Hydrogen sulphide producing test
- Decarboxylation
- Urea hemolysis test
- Nitrate reducing test
- Ammonia producing test
- Starch haemolysis test
- Lipid haemolysis test
Carbohydrates are biological molecules which are made up of carbon hydrogen and oxygen molecules. There are four types of carbohydrates and they are shown in the following
- Monosaccharides
- Disaccharides
- Oligosaccharide
- Polysaccharides
Carbohydrates and its types
Monosaccharides
Monosaccharides are simple molecules of sugar it is water soluble and crystallin in nature example are glucose propose and galactose.
Disaccharides
Disaccharides is a simple carbohydrate forms when two monosaccharides molecules are joined together and a water molecules is removed. example lactose sucrose and maltose.
Oligosaccharides
Oligosaccharide is a polymer containing small number of monosaccharides. example fructo- oligosaccharide galacto oligosaccharide and Mannan.
Polysaccharides
Polysaccharides are complex carbohydrate containing long chain of monosaccharides. example starch glycogen cellulose.
Carbohydrate fermentation test
Fermentation
Fermentation is a metabolism process in which the bacteria or yeast convert substance into a product like acid gas or alcohol. the product formed in fermentation process of economic value.
Carbohydrate fermentation tests demonstrate fomentation of sugar like glucose lactose or sucrose play Stop The fermentation is noted by production by bacterial cells
Requirement
- Nutrition broth medium
Glucose broth medium
Sucrose broth medium
Lactose broth medium
2. Indicator phenol red and andrades indicator.
3. Durham's test tube
Procedure different test tube containing three different type of sugar broth and invert a durham's tube in it and screw up to the test tube
Further the three sugar brought tube are sterilized by the autoclaving.
After sterilization the tubes are cool down to the room temperature and inoculated with the cell suspension in aseptic condition.
The tubes are incubated at 37°C for 24 hour
After incubated the tubes are examine for acid and gas production and result and note down.
Mechanism
The three style sugar broth tubes are incubated with bacterial Suspension and incubated for 24 hour. during incubation the bacterial cells may or may not utilise sugar.
If the cells utilise sugar Jacky is acid and gases production and this change is observed by a change in colour of broth.
First of all in sugar broth, phenol red indicator is added this PH sensitive indicator is added for determine of acid production initially.
The broth tubes are red in colour and after incubation if there is acid production the colour of broth changes from red to yellow. If there is no changes in colour of broth then there is no acid production.
Secondly in sugar broth durhman's test tube is inverted to determine gas production after incubation. If a gas bubble is observed and there is gas production.
Notes red is a PH sensitive indicator its pH range is at acidic pH it is yellow in colour at alkaline pH is red in colour.
Observation 1
There are three possibility and they are after incubation
1. if the red colour abroad is observed then there is no fermentation observation in diagram
In the above image a is incubated with a cell Suspension and incubated at 37°c per 24 hour and after incubation the red colour was not changed to yellow and this is incubated that there was no acid and gas production
Observation 2
In the above image test tube B is intubated with cell Suspension and incubated at 37°c for 24 hour and after incubation the colour of test tube change from red to yellow due to the acid production and there are no gas formation.
Observation 3
In the above image test tube C was incubated with cell Suspension and incubated at 37°C for 24 hours and after incubation the red colour of broth was changed to
Result
1. If there is no changes in colour of broth then the test is negative
2. if the yellow colour of broth is observed fomentation of carried out resulting in acid production and it is indicated by the positive sign
3. if the yellow colour of broth along with gas bubble accumulation is observed and fermentation is carried out resulting in acid and gas production
Indole production test
Aim. the aim of this test is to determine the capability of a bacterial culture to produce indole.
Requirement
Nichrome wire
Bacterial culture
Tryptophan Broth
Kovac's reagent enrich reagents
Xylene
Procedure
Tryptone broth is prepared distributed in the test tube and sterilized by autoclaving.
The Sterile trypton broth tubes are incubated with loopful of suspension and incubated at 37°C for 24 hour.
After incubation add 3 to 4 drops of xylene in tubes and shake vigorously and keep the tubes still so that two layers get separated.
After Separation of two layers add 1 ml of kovac's reagent aur enrich's reagent and tubes are observed for formation of pink colour ring.
Indole test is generally use of different between Escherichla specimen Enterococus specimen.
Observation
Red ring = positive indole test (E Coli)
Yellow ring= negative indole test ( Klebsklla)
Catalase test
Principal
Certain bacteria have an enzyme catalase which act on hydrogen peroxide to release oxygen
Procedure
Pick up a few colonies of test bacteria with plastinum loop from nutrient Agar slope (plate and mix it in a drop of H2O2 ( 10 volume) on a clean glass slide.
A positive catalyst reaction reduce gas trouble immediately. since blood contain catalyst culture on blood containing media may result in false positive reaction used of iron wireloop for picking up bacterial colonies maybe another cause of false positive test.
Interpretation
Positive test = immediately bubbling easily observed
Negative test = no bubbling
Positive and negative bacteria
Catalase positive . all members of intra bacteria cases expect shigella
dysenterioe type 1 staphylococcus micrococcus bacillus.
Catalase negative. Shigella dysenteriae type 1 Streptococcus clostridium catalase test is primarily used to differentiate between genera staphylococcus from Streptococcus.
@bacteria