To lay down the procedure for sub culturing of microorganisms from pure cultures and preparation of standardized viable culture suspension.
1.0 Purpose :
To lay down the procedure for sub culturing of microorganisms from pure cultures and preparation of standardized viable culture suspension.
2.0 Scope :
This procedure applies to preparation of, viable culture suspension, and sub culturing and maintenance of the following cultures for the given organisms against the media used and incubation conditions in microbiology laboratory.
Organism ATCC No. Media Used Incubation
Salmonella abony 6017 SCD agar 30 – 35ºC/24 hours
Pseudomonas aeruginosa 9027 SCD agar 30 – 35ºC/24 hours
Bacillus subtilis 6633 SCD agar 30 – 35ºC/24 hours
Escherichia coli 8739 SCD agar 30 – 35ºC/24 hours
Staphylococcus aureus 6538 SCD agar 30 – 35ºC/24 hours
Candida albicans 10231 SD agar 20 – 25ºC/48 hours
Clostridia sporogenes 11437 SCD agar / 30 – 35ºC/24 hours
Columbia agar
3.0 Responsibility:
The responsibility of sub culturing and preparation of viable culture suspension of the micro-organisms as per the procedure lies with the Microbiologist/Analyst.
4.0 Definition/Abbreviations: Nil.
5.0 Procedure :
5.1 Sub culturing:
5.1.1Pure cultures shall be procured from National Collection of Industrial Microorganisms (NCIM), Pune or any approved sources. Check for the confirmation of morphological characters. After conforming, transfer each organism into SCDM and incubate it for 24 hours and clostridia in Robert cooked meat medium, subculture each organism over seven agar slants under LAF(clostridia on SCD agar/Columbia agar slant). Or Take the required cultures from any of the Mylan Labs Units with traceable data subculture each organism over seven agar slants under LAF,
5.1.2Heat the Nichrome loop to the red-hot over the flame.
5.1.3Bring the broth of pure culture and freshly prepared slant near the flame.
5.1.4Remove the plug of the tubes by holding the culture tube between the little finger and ring finger and fresh slant between ring finger and middle finger of the left hand.
5.1.5Using right hand unplug the cotton of the pure culture by holding betweenmiddle finger and ring finger and fresh slant plug between ring finger and little finger.
5.1.6Heat the neck of the both tubes under flame.
5.1.7Charge the loop with pure culture by holding between index finger and thumb of the right hand.
5.1.8Transfer and streak the culture on the surface of the fresh slant to be sub- cultured, in a wavy pattern with crests and troughs.
5.1.9Flame the neck of both tubes and plug the cotton and ensure that plugs goes to the respective tubes. Follow same as above mentioned steps from 5.1.2 to 5.1.9 for the other cultures.
5.1.10Label them as SC-I, SC-II, ---SC-VII and keep SC-VII as reference.
5.1.11SC-I shall be used for 1st month followed by SC-II for 2nd month and so on to SC-VI for 6th month.
5.1.12For the first month prepare 4 working cultures from SC-I, and labels them as 1,2,3&4 Working culture-1 shall be used for 1st week, 2 for 2nd week and so on and all tubes are incubated as per Annexure-3
5.1.13After incubation store all the cultures at 2-8ºC.
5.1.14The above cultures shall be used with in 6 months.
5.1.15After 5th month initiate the procurement of fresh cultures.
5.1.16Each working cultures shall be destroyed after one week (Ex: SCI-1 shall be destroy after 7 days).
5.1.17Stock cultures (SCI, SCII,…so on) shall be destroyed after every month. Reference cultures (SCVII) and Source cultures (Any National Collection Center) shall be destroyed only after procurement of new cultures.
5.1.18A log book shall be maintained with the details as shown in Formatxxxx
5.2 Viable culture suspension:
5.2.1Take one loop full of culture from working culture slant and add it to 10ml sterile sodium chloride peptone solution and label it as stock solution.
5.2.2From the stock solution suspension, withdraw 1ml and transfer it into test tube containing 9mL sterile sodium chloride peptone solution. Mark this tube as – 1.
5.2.3Transfer 1ml from Tube as -1 to next tube containing 9 mL sterile sodium chloride peptone label this tube as -2 and so on do same as above process upto tube -10. Take 1mL from each dilutions of 10-5 to 10-8 as per the flow chart of serial dilution as shown in Annexure-2, into petri dishes in duplicate for trial and error knowledge of the organism and add 15–20ml of molted media and spread it uniformly under LAF. Incubate at appropriate temperature and duration as given in Annexure –3. Count and record the colonies of plated dilutions from 10-5 to 10-8 and make the average of the two plates, if colonies are more than 100 give the value as TNTC(Too Numerous To Count).
5.2.4Acceptance criteria: 10 - 100 CFU
5.2.5Count range from 10 - 100 CFU per plate shall be considered as valid and use this dilution for GPT testing or other purposes.
5.2.6If the count is not in the range suitable dilutions shall be made from 10-8 -10-12 dilutions. Repeat the same procedure as mentioned in 5.2.3 step until to get required CFU.
5.2.7The valid dilution shall be stored at 2-8ºC and valid for 9 days from the date of preparation. Destroy the residual suspension and stock suspension by autoclaving at 121ºC /15lbs for 30 minutes.
5.2.8Store the before dilution also (for example: 10-6 ) with valid dilution (for example: 10-7), which can be use for further dilutions. Transfer 1 mL from Ex:10-6 dilution in 9mL of sodium chloride peptone to get Ex:10-7 dilution in case of valid dilution is exhausted.
5.2.9After 9 days the dilution suspensions shall be destroyed by autoclaving at 121ºC /15 lbs/ for 30 minutes.
5.3 Alternatively use BioBall or Ready made culture suspensions instead of sub-culturing and preparation of viable culture suspensions.
5.3.1 Procure the BioBall(MultiShot-550) or Ready made culture suspensions from suitable vendor with Certificate Of Analysis of each culture suspensions as per current guideline ATCC No. /NCTC No..
5.3.2 Check the BioBall(MultiShot-550)or Ready made culture suspensions expiry date or its validity.
5.3.3 Re-hydrate the BioBall(MultiShot-550) vial with 1.1 mL of rehydration fluid before the use.The each BioBall having cells of microorganism which are suspended in fluid called them as culture suspension.
5.3.4 Each BioBall(MultiShot-550) culture suspension has mean between 500 and 600 cfu.
5.3.5 When rehydrated into 1.1 mL with rehydration fluid, each 100 µL or 0.1 mL of culture suspension having 50 cfu with standard deviation is less than 10%.
5.3.6 As per requirement the BioBall cultures are rehydrated.
5.3.7 Use for Pour plate method: Take 100 µL or 0.1 mL of culture suspension into sterile petriplates and pour the molted sterile agar media and incubate it as per requirements.
5.3.8 Use for Spread plate method: Take 100 µL or 0.1 mL of culture suspension on prepared agar media and spread it with sterilized spreader, use separate spreader for each activity and incubate it as per requirement.
5.3.9 Use for Broth media: Take 100 µL or 0.1 mL of culture suspension into sterile broth media and incubate it as per requirements.
5.3.10 The rehydrated culture suspensions can be stored at 2-8ºC for 8 hours after rehydration.
6.0 Related Documents: Nil.
Microbiology, sub culturing, microorganisms, pure cultures, viable cultur
