Identification of bacteria in microbiology

b pharm 2nd year 3rd sem

1. Identification of bacteria

2. Identification of bacteria Bacterial identification techniques can be broadly classified into two categories: Traditional and Modern methods. Traditional methods include: Macroscopic features: examining the overall appearance of a microorganism, including its shape, size, color, and smell. Microscopic features: observing the cell shape and staining characteristics such as gram status and cell structures. Culturing techniques: studying the growth characteristics such as oxygen, osmotic pressure, temperature requirements, and colony morphology. Biochemical tests: performing tests such as oxidase, catalase tests, and using selective/differential media. Modern methods include: Genotypic methods: genetic analysis through the use of nucleic acid probes or other molecular techniques. This includes polymerase chain reaction (PCR) which is widely used for detecting and identifying pathogens. Immunological methods: these methods use the immune response of the host to identify the bacteria. Mass spectrometry: this technique identifies bacteria based on their protein profile. Next-generation sequencing: this method provides a comprehensive view of the bacterial genome, allowing for precise identification.

3. Staining (Microscopical) techniques Staining techniques are used to enhance the contrast of microorganisms so that they can be visualized under a light microscope. Bacteria are typically colorless and transparent, making them difficult to see without staining. Type of staining:

4. Simple staining In simple staining is perform to the morphological characteristic by comparing the shape and size of bacteria. In simple staining different basic dye solution are used such as crystal violet, safranin, methylene blue. Simple staining is also called as monochrome staining or positive staining Principle Simple staining is a microscopic staining technique that uses a single dye to color all bacterial cells on a slide the same color. This technique is based on the principle of electrostatic attraction, whereby positively charged dye molecules are attracted to and bind to negatively charged components of bacterial cells, such as nucleic acids and cell walls. Procedure

5. Gram’s staining On the basis of there structure bacteria is classified into two class gram + ve and – ve . The differential staining technique by which gram+ve and gram– ve bacteria is identify is called gram staining. This staining technique was developed by Han’s Christian gram in 1884. Gram + Gram – In this peptidoglycan is present multilayer Single layer In this outer membrane is absent Outer membrane is present Cell wall 20-30nm thick and single layer Cell wall 5-12nm thick and two layer. After gram staining it gives purple/blue color. It gives Red/Pink color. Eg. Streptococcus, bacillus etc. Eg. Eschercheia coli  

6. Procedure The Gram staining procedure can be summarized in the following steps: Prepare a smear of the sample: A small amount of the sample is spread on a clean glass slide and allowed to air dry. Heat-fix the smear: The smear is passed through a flame to heat-fix the bacteria to the slide. Apply the crystal violet stain: The crystal violet stain is applied to the smear for 1 minute. Rinse with water: The smear is rinsed with water to remove excess crystal violet stain. Apply Gram's iodine: Gram's iodine is applied to the smear for 1 minute. Rinse with water: The smear is rinsed with water to remove excess Gram's iodine. Decolorize the smear: The smear is decolorized with ethanol or acetone for 10-20 seconds. Rinse with water: The smear is rinsed with water to remove excess decolorizer. Apply the safranin counterstain: The safranin counterstain is applied to the smear for 1 minute. Rinse with water: The smear is rinsed with water to remove excess safranin counterstain. Air dry the smear: The smear is allowed to air dry. Observe the smear under the microscope: The smear is examined under a microscope with oil immersion objective.

7. Acid Fast Staining 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. It is the differential staining techniques which was first developed by Ziehl and later on modified by Neelsen . So this method is also called Ziehl- Neelsen staining techniques. Neelsen in 1883 used Ziehl’s carbol -fuchsin and heat then decolorized with an acid alcohol, and counter stained with methylene blue. Thus Ziehl- Neelsen staining techniques was developed. Principle: Acid-fast staining is a microbiological technique used to differentiate acid-fast bacteria from non- acid-fast bacteria based on their cell wall characteristics. This staining method relies on the property of certain bacteria to resist decolorization by acid- alcohol (a mixture of hydrochloric acid and ethanol) due to the unique composition of their cell walls.

8. Application of Reagent Acid fast Non-acid fast Primary dye Carbon fuchsine Red Red Decolorizer Acid / alcohol Red Colorless Counter stain Methylene blue Red Blue

9. Biochemical test - IMV i C Indole Test Methyl Red Test Voges-Proskauer Test Citrate Utilization Test IMViC is a set of four biochemical tests that play a crucial role in microbiology. These tests are primarily used to identify and differentiate various groups of bacteria, with a particular focus on Enterobacteriaceae. This family of Gram-negative bacteria is commonly found in the intestinal tract of humans and animals. The IMViC tests are indispensable tools for the identification and classification of these bacteria.

10. Indole Test To check the ability of bacteria to degrade the amino acid Tryptophan, Many bacterial species which possess enzyme tryptophanase. Tryptophanase degrade amino acid tryptophan to indole, Pyruvic acid and ammonia. p- Dimethylaminobenzaldehyde ( Kovac’s reagent ) react with indole & produce Quinoidal red – violet compound. Procedure: Inoculate the test microorganism into peptone water for check indole production, incubating at 37 o C for 48-96 hours. (2-4 Days) Then and 0.5ml of Kovac’s reagent and mix gently. Result: A red colour layer formed which Indicates that positive reaction (Production of indole). E.coli, Enterococcus faecalis, Haemophilus influenzae give Indole test positive. Basillus , Pseudomonas, Klebsiella species give Indole test negative .

11. Methyl Red Test To check the ability of bacteria to perform Mixed acid fermentation of glucose into acetic acid, lactic acid & succinic acid. Due to production of acid, pH of the medium falls and it maintained below 4.5. Methyl red is add to the broth as indicator, medium turns red. Procedure: Inoculate the test micro-organism in glucose phosphate both then incubate at 37 o C for 2-5 Days. Then add few drops of 0.04% solution of methyl red, mix and absorb the result in the form of change in color. Result: Red colour signifies positive (MR test) No colour change (Yellow Colour ) signifies negative. If test is Positive then E.coli, Salmonella & Shigella species of bacteria. If negative then Kelbsiella & Enterobacter species of bacteria.

12. Voges-Proskauer test (VP test) To check the ability of bacteria to produce A cetylmethylcarbinol (Acetoin) - a non acetic neutral end product of glucose fermentation. Glucose fermentation leads to the production of Acetoin. Acetoin is converted into diacetyl in the presence of α -naphthol, 40% Potassium hydroxide ( Barritt’s reagent A & B) & atmospheric oxygen. The diacetyl and guanidine containing compounds founds in the peptones of the broth then condense to form a pinkish red polymer. To check whether the microorganism follow 2,3 butanediol production pathway for glucose metabolism or not. VP positive ( Pinkish red color at the surface ) organisms include Klebsiella & Enterobacter species VP negative ( whitish yellow) organisms include E. coli, Streptococcus & Salmonella species.

13. Citrate Utilization Test In this test check the use of citrate as a carbon source for growth. Koser’s Citrate Medium has citrate as the source of carbon. It is used to differentiate Escherichia coli from Enterobacter aerogenes on the basis of citrate utilization. Ability of micro-organism to use citrate as the carbon source is indicated by the production of turbidity in the medium. Result: Positive – medium will be intense Prussian blue, growth of organism will be visible (Enterobacter aerogenes, Klebsiella species) Negative – No growth, medium remain in Green colour (Escherichia coli, Salmonella species)