Microbiological laboratories are involved primarily in culturing, examination and identification of microorganisms. These processes involve a number of techniques that a microbiologist should be well acquainted with.
The basic techniques practiced in a microbiological laboratory are
1.Aseptic techniques and Sterilization:
Autoclave is the instrument used in the microbiology labs for sterilization of all types of media and solutions. At 121°C and pressure 15 lb/sq. in. for 15 minutes, all living organisms including spores are killed in an autoclave.
All the apparatus and glassware are sterilized in a hot air oven at 160 °C for 2 hrs or exposure to radiation.
Aseptic techniques are employed to minimize the chances of bacterial contamination. This is accomplished by disinfection of working areas with disinfectants such as phenols, alcohol, surfactants, detergents, halogens etc., minimising possible access by bacteria from the air to exposed media, and use of flames to kill bacteria while working on cultures and inoculation.
2.Preparation of Microbiological media
Microorganisms have specific nutritional requirement as far as growth is concerned. The two basic media used in bacteriology are
- Nutrient broth, which is a liquid medium used to culture bacteria usually in tubes or conical flasks.
- Nutrient agar which is set into a jelly by the addition of a seaweed extract called agar, and the melt on heating is poured into glass or plastic petri dishes or plates.
Different types of media are used for specific microorganisms.
3.Inoculation and Incubation:
To prepare a culture, the bacteria may be introduced to the media (inoculated) by various means.
- A heat sterilized inoculating loop is usually used to introduce a small amount of any microbial culture in a liquid medium (broth)
- On a solid media the inoculation is done using a heat sterilized inoculating loop or a micropipette using sterilized tips.
- The techniques include pour plate method, spread plate method and the streak plate method.
The liquid broth or the solid agar media (in petri plates) are incubated i.e. placed in incubators that have a regulated temperature (usually 37°C for bacteria and 25-28°C for yeast and molds). The petri plates with bacterial inoculation are kept in inverted positions to prevent condensation droplets from falling onto the surface of the agar. Petri dishes are kept sealed with paraffin strips to prevent any contamination and maintenance of the cultures for a longer time.
Petri dishes can be stacked and conveniently stored in the refrigerator
4.Enumeration of Bacteria
Enumeration of microorganisms in a given sample is an important aspect of microbiology. There are different methods employed for the enumeration of bacteria in a given sample.
- Standard (Viable) Plate Count: in this method the samples are serially diluted, plated on an agar plate and incubated for growth of the colonies. Total number of colonies that grow are counted and multiplied with the dilution factor for obtaining the number of bacteria present in the sample.
- Direct Microscopic Count: In this method a ruled slide consisting of a counting chamber is used to directly count the number of microbes present in a sample. Example – Petroff- Hausser counting chamber.
- Turbidimetric measurement: As the bacteria start growing in a liquid medium after inoculation, the media becomes turbid and dense. This can be measured using a spectrophotometer which allows/ blocks the passage of light of a given wavelength.
5.Identification of Microorganisms
Accurate identification of microorganism is very essential in wide ranged applications of microbiology. Identification of microorganism is done through various methods
- Study of morphological characteristics are useful in basic categorization of any microorganism.
- Staining is a very important method in identification if microorganisms. Gram staining and acid fast stains are the basic staining methods used in any microbiology laboratory.
- Microscopes are used to observe microorganisms after all types of staining.Living bacteria can also be detected by direct observation using a light microscope.
- Biochemical tests are widely employed for the identification of bacteria. These tests exploit the metabolic properties of the microorganisms for their identification.
- Serological tests as slide agglutination, ELISA, and Western blotting,that involve the reactions of microorganisms with specific antibodies, are useful in determining the identity of strains and species.
- The molecular methods for bacterial identification include % G+C comparisons, PCR, rRNA sequencing, DNA fingerprinting by restriction fragment length polymorphism, or RFLP, nucleic acid hybridization.
Points to remember:
- In a microbiology lab, autoclave is used for sterilization of all media or solutions. Glass wares are sterilized in hot air ovens
- Nutrient broth and nutrient agar are the most common media used for bacterial growth.
- Pour plate, spread plate, streak plate methods are employed for obtaining pure cultures
- The growth temperature for bacteria is 37°C and 25-28°C for yeast and molds.
- Number of bacteria in a given sample can be obtained by plate count, direct microscopic count or spectrophotometric method.
- Staining is the most primary step in the identification of bacteria
- Biochemical tests serve as a major platform for bacterial identification.
- Serological and molecular methods are used for confirming the microorganism identification to the generic and species level.