Getting Started with an ATCC Bacterial Strain

Bacterial Culture Guide Getting Started with an ATCC Bacterial Strain


Table of Contents

Getting Started

ATCC bacterial strains are shipped frozen on dry ice in plastic cryopreservation vials, as lyophilized cultures in glass ampoules or serum vials, or as live cultures on agar slants or in broth medium. Upon receipt of frozen cultures, immediately revive cultures by thawing and subsequently transferring cultures to an appropriate growth medium. If this is not possible, store frozen vials in liquid nitrogen vapor (below -130°C). Alternatively, the frozen material can be stored between -70°C and -80°C for short periods (1 to 5 days); however, viability will decline at temperatures above -130°C. Freeze-dried cultures can be stored safely at 4°C or lower. Upon receipt, rehydrate or dilute cultures using the appropriate medium and incubation conditions specified on the product sheet. Live cultures should be transferred to an appropriate growth medium upon receipt and incubated under the conditions specified on the product sheet.

Product Sheet

ATCC bacterial strains are shipped with a product sheet that contains detailed information on the initiation and expansion of materials, as well as ideal growth and propagation conditions. This product sheet, as well as additional information, can be found on the ATCC website or can be requested from the ATCC Technical Service Department.

Nomenclature Changes

Changes in taxonomy or further analysis of bacterial strains may lead to a change in nomenclature. These changes are indicated on product sheets and can be found online on the product page. Further information on bacterial nomenclature changes can be found on the List of Prokaryotic Names with Standing inNomenclature (LPSN).

Preparation of Medium

In advance, prepare the appropriate medium and additional reagents necessary for bacterial strain revival and growth. Ensure that the proper incubation conditions are ready. Information for the formulation and preparation of the media and incubation conditions for these products is available on the ATCC website.

Opening Glass Ampoules

Overview

All cultures should be considered potentially hazardous and should be opened by individuals trained in microbiological techniques working in facilities with containment requirements appropriate for the biosafety level of the cultures. ATCC recommends that the handling or opening of glass ampoules be performed in a biological safety cabinet. If this is not possible, wear protective clothing, gloves, a face shield or safety goggles, and hold the vial away from your body. Ensure that all empty vials are sterilized before disposal.

Glass Ampoules

Opening Double-Vial Preparations (see diagram below)

  1. Heat the tip of the outer vial in a flame.
  2. Add a few drops of water on the hot tip to crack the glass.
  3. Strike the end of the vial with a file or pencil to remove the tip.
  4. Remove the insulation and inner vial with sterile forceps. Gently raise the cotton plug.

Opening Single-Vial Preparations (see diagram below)

  1. To recover the cell suspension from the glass ampoule, score the neck of the ampoule with a small, sterile file.
  2. Disinfect the outside of the ampoule with freshly prepared 70% ethanol or dip it into a beaker of freshly prepared 70% ethanol. 
  3. Wrap the ampoule within several folds of a sterile towel or gauze to dry residual ethanol. 
  4. Working in a laminar flow hood, hold the vial upright and snap open the vial. Ensure that your gauze does not become too wet with ethanol, or alcohol could be sucked into the culture when the vacuum is broken. Rehydrate the material immediately
Ampoules instructions

Initiating Frozen Cultures

  1. Prepare a sterile test tube that contains the recommended medium for bacterial growth as listed in the supplied product sheet. Ensure that the medium contains all necessary reagents and is equilibrated for temperature and pH. 
  2. Thaw the sample vial via gentle agitation in a water bath that is set to the normal growth temperature of that strain. Thawing will be rapid; approximately 2 minutes or until all ice crystals have melted. 
  3. Remove the vial from the water bath and decontaminate the outer surface using 70% ethanol. Follow strict aseptic conditions in a laminar flow hood for all further manipulations. 
  4. Unscrew the top of the vial and transfer the entire contents to a sterile test tube containing the appropriate growth medium. Additional test tubes can be inoculated by transferring 0.5 mL of the primary culture to additional secondary cultures. 
  5. Incubate cultures under the appropriate temperature and atmospheric conditions as recommended on the product sheet. 
  6. Examine cultures after the recommended incubation period. The incubation period will vary between strains and is listed on the product sheet. (See: NOTE 1)

NOTE 1:

Following recovery from cryopreservation or lyophilization, some bacterial strains may exhibit a prolonged lag phase. These strains may require an extended incubation period.

Initiating Lyophilized Cultures

  1. Using a Pasteur pipette, aseptically add 0.5 mL of the recommended growth medium to the freeze-dried material. Mix well. 
  2. Transfer the entire suspension to a test tube containing 5 to 6 mL of the recommended medium. Additionally, transfer several drops of the suspension to an agar slant. 
  3. Incubate cultures under the appropriate temperature and atmospheric conditions as recommended on the product sheet. Additional test tubes can be inoculated by transferring 0.5 mL of the primary culture to additional secondary cultures. 
  4. Examine cultures after the recommended incubation period. The incubation period will vary between strains and is listed on the product sheet. Most freeze-dried cultures will grow within a few days. (See: NOTE 1)

Handling Test Tube Cultures

  1. Incubate the culture upon receipt under the appropriate temperature and atmospheric conditions recommended on the product sheet. Do not store the culture in a refrigerator. 
  2. Transfer the culture to fresh media as specified on the product sheet. When transferring a broth culture, aseptically withdraw approximately 1.0 mL of the culture and transfer into 5 mL of fresh broth, or transfer several drops of the suspension to an agar slant or plate. When transferring a culture from an agar slant, aseptically transfer a single colony to 5 mL of fresh broth or to an agar slant or plate.
  3. Incubate the culture under the appropriate temperature and atmospheric conditions recommended on the product sheet.
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