ASEPTIC TECHNIQUE

ASEPTIC TECHNIQUE

     - Are the technique used by biologist to taking all prudent precautions to prevent contamination of culture and spread Micro-organism into the environment
- Aseptic technique are procedure that allow us to handle culture and sterile culture media without introducing microbial contaminants from air, water, hands, mouth and other sterile source
- It help in avoiding any contact between the sample or pure culture medium and sterile surface so disinfectant used to reduce changes of potential contaminants
- Sterilization of instruments to avoid chances and used to trnsfer bacterial culture in following ways
i) Flame the transfer loop until dried
ii) Uncap the tube
iii) Briefly flame the mouth of the tube
iv) Culture growth is picked up on the fresh broth or agar slant
v) Reflame the inoculating loop and recap the culture tube microbial cell/conidia also transfer surface of agar tube
@ Isolation of pure culture :-
       Different method of playing for getting pure culture are
1. Streak Plate method:-
        -Developed by Loeffler and Craffkey in lab of Robert Koch,
-this separation depend upon spatial separation of single cell
- In this mixed microbial culture is transferred to the edge of agar plate and then series of parellel non-overlapping streaks with specific pattern
- As microbes are rubbed off the loop to the medium, These is a continues reduction in microbes till last cell rubbed off
- Incubating loop streak can be of the type
* T-streak
* Quardrant streak
* Continues /full plate and etc
# Advantage :-
    This method was well when the organism to be isolated the present in large amount in a mixture
# Limitations :-
     This method is has suitable of Micro-organism which are to gone and broth
# Precautions :-
i) Avoid pressing the loop two formely against agar
ii) Maintain aseptic condition
2. Pour plate method :-
           In this method mixing small volume of microbial suspension with molten nutrient agar at 45° C and pouring immediately into sterile petric plate.
- This method involves adding specified amount of the dilution to the sterile petri plate
- Colonies appeared within the nutrient agar, as well as on surface of agar plate
- This method used in microbial cell enumeration in the original shape
# Advantage :-
            Colonies grow both on the surface and beneath the agar surface, so aerobes facultative anaerobes and non-stringent anaerobes can be studied
# Disadvantage :-
    Heat sensitive Micro-organism may be damaged by melted agar giving like viable count as compared to spread plate
3. Spread Plate method :-
         In this method a small volume of the dilute sample is transferred to the centre of a pre-poured solidified agar plate and spread uniformly spread on surface of the medium with spreader
# Advantage :-
      Useful for the sample having heat sensitive mictobes
# Disadvantage :-
          Volume no greater than 0.1 ml can be spread on nutrient agar plate b'coz it would not soaken well and may result colonies to cools as they form
@ Serial dilution agar plate method :-        In this method 1ml of sample is transferred to 9ml the dilution to get 1:10 dilution ,1 ml of this is transferred to another tube of 9ml of dilute to get 1:100 dilutions and so on required dilution are obtained
- Many biologist use plate with 30-300 colonies to get the microbial number in the sample plates , Plate getting colonies less than 30 are used marked to less to count TLTC ( Two less to count)  and getting more then 300 are marked as too numerous to count (TNTC) and are discarded
# Advantage :-
- It provide viable count as only live cell will grow and produce the colony
- Even very few cell in a sample can be counted
- Allow isolated of discrete colonies which can be sub-cultured to achieve pure culture
# Disadvantage :-
- Time consuming in this is usually 24 hour
- Microbial count obtain depends upon the culture media used for plate count
- More glassware needed