CDST_LT: Storage of culture isolates

Maintenance and Storage of Mycobacterial Strains

Strains may be well-characterized clinical isolates or culture collection strains (e.g. American Type Culture Collection, ATCC). Mycobacterial strains must be stored in adequate conditions to preserve their viability and intact genetic background. Maintenance of mycobacterial cultures by rre-inoculatingmedia during prolonged time may cause accumulation of mutations that can lead to unpredictable results. 

 

Steps to preserve and store MTB cultures 

 

1. From culture on solid media

1. Prepare suspension equal to McFarland 1.0 standard from a culture not older than two weeks after growth appearance.

2. Label sterile screw-capped cryovials with laboratory number, study reference and date of preparation

3. If suspensions are from quality control strains, label them with name, ATCC number, batch number and date of preparation.

4. Distribute the suspension into sterile screw-capped cryovials.

5. Put the cryovials into a numbered storage box. 

6. Put the storage box into the -80C freezer.

7. Record the location and strain identifiers in the -80°C Freezer Storage Logbook.

    

   2. From liquid culture

8. Label sterile screw-capped cryovials with laboratory number, study reference and date of preparation

9. If cultures are from quality control strains, label them with name, ATCC number, batch number and date of preparation.

10. Distribute the suspension into sterile screw-capped cryovials.

11. Put the cryovials into a numbered storage box. 

12. Put the storage box into the -80°C freezer.

13. Record the location and strain identifiers in the -80°C Freezer Storage Logbook.

     

    Storage

     

    Short-term storage

• Cultures on solid media can be stored at room temperature in the dark for several months and 4°C  for two years.

• Liquid cultures can be maintained viable at 4°C  for several months. 

   

   Long-term storage

• Cultures can be stored at -20°C  for several years and at -80°C  for decades.

   

   

  Resource

   

Mycobacteriology Laboratory Manual 

Assessment

 

• Question​	Answer 1​	Answer 2​	Answer 3​	Answer 4​	Correct answer​	Correct explanation​	Page id​	Part of Pre-test​	Part of Post-te
  Cultures can be stored for how long?	-20°C  for several years	-80°C  for decades.	Answer 1	Answer 2	Both Answer 3& 4	Cultures can be stored at -20°C  for several years and at -80°C  for decades.

   

   

       

 MTB isolates from all positive cultures (using the LJ subculture from an MGIT tube positive for MTB or a subculture from the original LJ culture, if MGIT is unavailable) will be frozen in 7H9 broth and glycerol to preserve them for any repeat or additional microbiology tests that may need to be performed (either in-house or at a central laboratory). It is recommended that at least four aliquots from baseline visits, and two aliquots from subsequent visits, be frozen for each MTB isolate.

 

• Media and consumables required to store culture isolates

  1. Positive LJ slants (Solid Culture: Lowenstein Jensen (LJ) Media)

   2.2 ml sterile cryotubes with a screw top, externally threaded

  3. Water bath

  4.7H9 media with glycerol

  5. ADC or OADC enrichment broth

  6. Sterile transfer pipettes with graduations marking volume (individually wrapped)

  7. Sterile loop, disposable applicator stick, or cotton swab

  8. Permanent marker

  9. Cryobox and rack

  10. Study-specific labels

     Resource

    https://stoptb.org/wg/gli/assets/documents/gli_mycobacteriology_lab_manual_web.pdf        

     Assessment

 

Question​	Answer 1​	Answer 2​	Answer 3​	Answer 4​	Correct answer​	Correct explanation​	Page id​	Part of Pre-test​	Part of Post-te
Glycerol is essential for storing culture.	True	False			True	Glycerol stabilises the frozen bacteria, preventing damage to the cell membranes and keeping the cells alive.

•  

 

Liquid media are used in the mycobacteriology laboratory for sub-culturing stock strains for storing strains at - 80°C and other in-vitro tests. Mycobacteria tend to clump in a liquid medium. Therefore,  a wetting agent like Tween 80 must be included in the basal medium. It encourages more homogenous growth. Commercially available Middlebrook 7H9 media, along with growth supplement OADC, can be utilized for this purpose.

Steps in Preparation of Selective Middlebrook- 7H9 Liquid Medium

Salt solution: Disodium anhydrous hydrogen phosphate (Na₂HPO₄): 2. 5g 

                      Potassium dihydrogen orthophosphate (KH₂PO₄): 1. 0 g 

                      Ammonium sulphate ( NH4SO4) : 0. 5g 

                      L-sodium glutamate : 0. 5g 

                     Trisodium citrate (2H2O) : 0. 1g 

                      Pyridoxine hydrochloride: 1. 0 ml of 0. 1% aq. soln. 

                      Biotin : 1. 0 ml of 0. 05% aq. soln. 

                      Ferric ammonium citrate (green) : 0. 5 ml of 8% aq. solution 

                      Magnesium sulphate (MgSO4.7H2O): 1. 0 ml of 5% aq. solution 

                      Calcium chloride (CaCl2.2H2O): 1. 0 ml of 0. 05% aq. solution 

                     Zinc sulphate (ZnSO4.7H2O): 1. 0 ml of 0. 1% aq. solution 

                     Cupric sulphate (CuSO4.5H2O): 1. 0 ml of 0. 1% aq. solution

                     Tween-80, 10% (For obtaining dispersed cultures): 5. 0 ml (Or) Glycerol: 5. 0 ml Distilled water to 900 ml.

                     Mix well, distribute in 95 ml amounts and sterilize at 15 lbs/15 mins.

The salt solution can also be prepared by using Difco dehydrated powder.

Weigh 4. 7 g of the dehydrated base into a 2-litre flask, and add 900 ml distilled water and 0. 5 ml of Tween-80 or Glycerol. Mix well, distribute in 95 ml amounts and autoclave at 15 lbs/15 minutes.

Before use, to each 95 ml salt solution, aseptically add 5 ml sterile ADC (bovine albumin-dextrose-catalase) solution and mix well.

Distribute in 5-10 ml amounts in sterile universal containers, check sterility by overnight incubation at 37 ^oC and store in the cold.

ADC supplement

Bovine albumin, Fraction V: 10g. 

Glucose, A. R. (dextrose): 4g. 

Catalase : 3 mg* 

*Dissolve 30 mg catalase in 10 ml water by vigorous shaking and add 1 ml of this solution. 

Distilled water: 100ml.

Mix well and sterilize by Seitz filtration or membrane filtration.

Resource

      STANDARD OPERATING PROCEDURE FOR MYCOBACTERIOLOGY LABORATORY

•       Assessment

 

Question​	Answer 1​	Answer 2​	Answer 3​	Answer 4​	Correct answer​	Correct explanation​	Page id​	Part of Pre-test​	Part of Post-te
Liquid media are used for which of the following?	Sub-culturing stock strains for storing strains at - 80°C	To perform  in-vitro tests	Only option 1	Both options 1 & 2	Both options 1 & 2	Liquid media are used in the mycobacteriology laboratory for sub-culturing stock strains for storing strains at - 80°C and other in-vitro tests.

 

 

Quality control (QC) Testing of 7H9 Liquid Medium

Every new lot of MGIT medium and every new lot of the enrichment should be quality control tested by the user upon receipt and before it is used routinely.

A. QC strains

Cultures: The following three mycobacterial cultures are recommended for quality control testing.

1.M. tuberculosis, H37Rv ATCC 27294
2.M. kansasii ATCC 12478
3.M. fortuitum ATCC 6841

 If the ATCC reference strains of M. kansasii or M. fortuitum cannot be obtained, then a laboratory isolate which is well-characterized may be used as a quality control strain. Well-characterized strains will be available from the mycobacterial strain bank of TDR/WHO in late 2006⁷³

B. Preparation of culture suspension

1.  Subculture the above mycobacteria on several LJ slants.
      • Incubate at 37ºC + 1ºC.
      • Observe growth visually.
      • As soon as there is good, confluent and pure growth, use this growth for making suspension. Growth should appear within 10-15 days of subculturing and should be used within this period. Aged cultures would not give reliable results.
2. Remove growth from the slant by carefully scraping the colonies off the slant with a sterile loop or sterile spatula made from wooden applicator sticks. Take extreme precautions not to scrape off any culture medium (which gives false turbidity measurement).
3.  Transfer growth into a screw cap tube containing 4 ml of sterile 7H9 broth and glass beads (6-10 beads, 1-2 mm diameter), which helps to break up clumps (Tube A).
4.   Vortex the tube for at least 1-2 minutes. Ensure the suspension is well dispensed and turbid (greater than McFarland #1 turbidity).
      Let the suspension stand undisturbed for 20 minutes.
5.    Using a transfer pipette, carefully transfer the supernatant to another sterile screw cap glass tube (Tube B). Avoid picking up any sediment.
      Let this stand undisturbed for 15 minutes.
6. Carefully transfer the supernatant into another screw cap glass tube (Tube C) without taking any sediment. Next, adjust the turbidity of suspension in Tube C to McFarland #0.5 turbidity standard by adding more 7H9 broth or sterile saline/deionized water and mixing well. If the suspension is too turbid, transfer some of the suspension to another sterile tube and adjust the turbidity to McFarland #0.5 standard.   This is the working suspension for QC testing. This suspension may be frozen in small aliquots (1-2 ml) in appropriate tubes/vials at -70ºC + 10ºC. The frozen suspension may be used for up to 6 months. Once thawed, do not refreeze.

 C.Preparation of dilutions

1. Dilute the working suspension McFarland #0.5, freshly prepared or frozen) 1:5 by taking 1.0 ml of suspension and adding into 4.0 ml of sterile water or saline. Mixwell (Tube 1).
2. Dilute two more times 1:10 by adding 0.5 ml of suspension Tube 1 into 4.5 ml of sterile water or saline (Tube 2). Mix well and then again add 0.5 ml from Tube 2 to 4.5 ml of sterile saline or distilled/deionized water (Tube 3). Mix well. Final dilution 1:500 (Tube 3). Stop here for M. tuberculosis and use Tube 3 for QC testing.
   • For M. fortuitum, further dilute Tube 3 1:10. Take 0.5 ml of suspension from Tube 3 and add to 4.5 ml of sterile water or saline and mix well. Final dilution 1:5000 (Tube 4). Use Tube 4 for QC testing.
   • For M. kansasii, dilute Tube 4 once again 1:10 by adding 0.5 ml from Tube 4 to 4.5 ml of sterile saline/water, mix well. Final dilution 1:50,000 (Tube    Use Tube 5 for QC testing.

 D. Inoculation/incubation
1. Supplement MGIT medium with Growth Supplement and PANTA as recommended.
2. Inoculate 0.5 ml from Tube 3 to each of the two MGIT tubes for M. tuberculosis. Similarly, inoculate 0.5 ml from Tube 4 for M. fortuitum and Tube 5 for M. kansasii into each of the two labelled MGIT tubes. Mix.
3. Enter the inoculated tubes in the MGIT 960 instrument. Take the tubes out when indicated positive by the instrument. Retrieve data for time to positive.

E. Expected results

 M. tuberculosis Tube fluorescence positive in 6 to 10 days
 M. kansasii Tube fluorescence positive in 7 to 11 days
 M. fortuitum Tube fluorescence positive in 1 to 3 days

If the above criteria are not met, repeat the test. If the QC test still does not give satisfactory results, check the viability of the inoculum, the age of the culture if stored frozen and other procedures. If everything meets the established specifications, contact Technical Services at BD Diagnostic Systems.

F. Precautions.

1. Use freshly prepared suspension adjusted to McFarland #0.5 standard. If frozen (-70ºC + 5ºC), thaw prior to use for each quality control testing. Do not store or refreeze once thawed.
2. All work should be carried out in a proper biological safety cabinet.
3. All materials should be sterilized by autoclaving prior to disposal.
4. Follow all the recommended safety precautions.

Resource

Mycobacteriology Laboratory Manual 

Assessment

Question​	Answer 1​	Answer 2​	Answer 3​	Answer 4​	Correct answer​	Correct explanation​	Page id​	Part of Pre-test​	Part of Post-te
Which of the following mycobacterial cultures are recommended for quality control testing?	M. tuberculosis	M. kansasii	M. fortuitum	All of the above	Answer 4	The following three mycobacterial cultures are recommended for quality control testing. 1.M. tuberculosis, H37Rv ATCC 27294 2.M. kansasii ATCC 12478 3.M. fortuitum ATCC 6841

 

 

   Steps to Preserve and Store MTB Cultures 

    

    From culture on solid media

1. Store Mycobacteria in the 7H9 Liquid medium.
2. Add 0. 5 ml of double sterile distilled water in a Bijou bottle containing glass beads (3 mm. diameter).
3. Add bacterial growth from one slope of LJ medium with 3+ growth, and mix using vortex mixture for about 30 seconds. Allow the coarse particles to settle down.
4.  Pipette out 100 μl of bacterial suspension into McCartney bottles containing five ml of 7H9 medium and Incubate at 37° C for 10 days.
5. After 10 days, examine for any gross contamination (turbidity). If turbidity is observed, discard the subculture and attempt a fresh subculture.
6. Inoculate a loopful of media on a nutrient agar plate.
7. Incubate at 37°C for 24 hours and check for sterility.
8.  Record the observations are recorded in the Sterility Check Register.
9. Using a 1ml tuberculin syringe, prepare uniform suspension by aspirating and discharging the suspension for about 10 times within the vial.
10. Adjust the turbidity to McFarland No.1 standard.
11. Pipette out aseptically 750 μl of bacterial suspension into a cryovial. Add 750 μl of 40% sterile glycerol.
12. Label it with the lab number & date and arrange it in a cryovial rack.
13. Store Cryovials at –80 C freezer.
14.  Maintain a date-wise list of all cultures stored with particulars of the culture in the storage register.

 

    From liquid culture

• Label sterile screw-capped cryovials with laboratory number, study reference and date of preparation.
• If cultures are from quality control strains, label them with name, ATCC number, batch number and date of preparation.
• Distribute the suspension into sterile screw-capped cryovials.
• Put the cryovials into a numbered storage box. 
• Put the storage box into the -80C freezer.
• Record the location and strain identifiers in the -80°C Freezer Storage Logbook.

Resource

 

Mycobacteriology Laboratory Manual 

Assessment

 

Question​	Answer 1​	Answer 2​	Answer 3​	Answer 4​	Correct answer​	Correct explanation​	Page id​	Part of Pre-test​	Part of Post-te
McFarland No.1 standard is used to adjust the turbidity. McFarland's	True	False			Answer 1 True	Culture isolates are adjusted to   McFarland No.1 standard to carry out the uniform suspension.

        

A sterility check of growth is ascertained to differentiate M. tuberculosis from other contaminants in MGIT-positive tubes. 

BHI (Brain Heart Infusion) Agar plates are used for sterility checks using the following steps:

1. Divide the plate into 4 quadrants so that four specimens can be subcultured onto one plate. 

2. Label the plate with specimen ID

3. Vortex the MGIT tube well, unscrew the tube cap and sample an aliquot of broth using a sterile, disposable pipette/inoculation loop. Remove about 200 μl of broth (~ 4 drops) and inoculate the BHI agar.

4. Incubate the agar plate in the incubator at 37 °C  for 72 hours.

Results:

1. Growth in 24 – 48 hours (Contamination)​
2. No Growth (Contamination free) ​

Resource

Mycobacteriology Laboratory Manual

Assessment

 

Question​	Answer 1​	Answer 2​	Answer 3​	Answer 4​	Correct answer​	Correct explanation​	Page id​	Part of Pre-test​	Part of Post-te
What does growth in 24 – 48 hours in BHI Agar indicate?	Contamination	M. tuberculosis complex detected	M. tuberculosis complex not detected	NTM detected	1	Growth in 24 – 48 hours in BHI Agar indicates contamination.		Yes	Yes

 

 

 

The MGIT system automatically and continuously incubates and monitors tubes once they are placed in a station; there is no need to remove the tubes from the instrument. 

Cultures during incubation:

1. Cultures remain in their stations until signalled “positive”, or if no growth is detected after 42 days incubation, are signalled “negative”. 

2. If an instrument-positive tube is determined to be smear-negative for either mycobacteria or contaminants, the tube may be re-entered into the instrument, but within 5 hours of removal.

3. If the tube is not returned within the 5-hour re-entry window, the associated data is removed from the instrument’s database, and the tube will be monitored as a newly entered tube.

The MGIT instrument will record the date each tube was entered.
• Do not turn tubes after placing them in the station.
• Do not remove tubes unless they are positive or out-of-protocol negatives (negative at 42 days).
• Do not reassign tubes to a new station.

Resource

Mycobacteriology Laboratory Manual

Assessment

 

Question​	Answer 1​	Answer 2​	Answer 3​	Answer 4​	Correct answer​	Correct explanation​	Page id​	Part of Pre-test​	Part of Post-te
What happens If the MGIT tube is not returned within the 5-hour window?	The tube can be returned without loss of data.	The test has to be assigned to a new station.	Data is removed from the instrument’s database, and the tube will be monitored as a newly entered tube.	The tube has to be discarded.	3	If the tube is not returned within the 5-hour re-entry window, the associated data is removed from the instrument’s database, and the tube will be monitored as a newly entered tube.

        

 

 

Revival of the frozen cultures 

Take the cryo vial with culture from the – 80°C freezer and place it in an ice bucket. Wipe off the outer surface with a 70% ethanol cotton swab and allow thawing on ice.

Inoculate a loopful of culture aseptically onto LJ media slopes. Then, incubate the inoculated LJ slopes for 4 weeks at 37°C. 

Store the cryo vial with culture back at -80°C 

Note: Repeated freeze and thaw will affect the viability of the stored cultures

Resource

Revised National TB Control Programme     

Assessment

 

Question​	Answer 1​	Answer 2​	Answer 3​	Answer 4​	Correct answer​	Correct explanation​	Page id​	Part of Pre-test​	Part of Post-te
Revival of frozen cultures is done by which of the following methods?	Thawing	Heating	Boiling	Spinning	Answer 1 (Thawing)	Revival of frozen cultures is done by thawing on ice.