Bacterial Growth and Propagation

Temperature

Because bacteria can grow and thrive in a variety of environments, optimal growth temperatures may vary significantly between species. In general, most pathogenic or commensal bacterial strains grow well at body temperature (37°C). In contrast, many environmental strains thrive at lower temperatures, often within a range of 25°C to 30°C.

Atmosphere

In addition to varying requirements for optimal growth temperatures, bacteria also differ in their use of oxygen for respiration. Aerobic organisms, such as Bacillus species, use oxygen as a terminal electron acceptor during respiration. Similarly, microaerophiles, such as Helicobacter pylori, also require the use of oxygen, but at lower levels than naturally occurring in the environment. In contrast, anaerobic organisms use electron acceptors such as nitrate or sulfate, among other inorganic acceptors. These inorganic compounds, however, have a lower reduction potential than oxygen thus resulting in less efficient respiration.

The use of oxygen and inorganic compounds by anaerobic organisms can differ greatly between species. Obligate anaerobes, such as Clostridium species, can only survive and reproduce in the absence of oxygen; these organisms are often killed by the presence of oxygen. Similarly, aerotolerant anaerobes, such as Lactobacillus species, cannot use oxygen during respiration; however, unlike strict anaerobes, these microorganisms can tolerate oxygen for short periods of time. Lastly, facultative anaerobes, such as Escherichia coli and Staphylococcus species, are able to survive in both the presence and absence of oxygen. If given the choice, these organisms prefer the use of oxygen during respiration as it has the greatest reduction potential as compared to other electron acceptors.

When working with anaerobic cultures, it is important to avoid unnecessary exposure to oxygen. Anaerobic conditions may be obtained for either transfer or incubation by the methods listed below. Unless specifically mentioned, the standard anaerobic gas mixture is 80% N₂, 10% CO₂, and 10% H₂.

Anaerobic conditions for transfer may be obtained by either of the following:

1. Use of an anaerobic gas chamber. 
2. Placement of test tubes under a gassing cannula system hooked to anaerobic gas.

During incubation, anaerobic conditions may be maintained by any of the following:

1. Loose screw-caps on test tubes in an anaerobic chamber. 
2. Loose screw-caps on test tubes in an activated anaerobic GasPak™ system.
3. Use of sterile butyl rubber stoppers on test tubes so that an anaerobic gas headspace is retained.

Propagation of bacterial strains can vary significantly between species. Below, we describe the general procedure for the propagation of non-fastidious strains and a more detailed set of procedures for fastidious strains and bacteriophages.⁴ Information regarding the recommended medium and growth conditions can be found on the supplied product sheet. The product sheet, as well as additional information, can be found on the ATCC website or can be requested from the ATCC Technical Service Department. Additional propagation information can also be found in Bergey’s Manual of Systematic Bacteriology 2^nd Edition, Published by Springer, New York.¹

Propagation of Non-Fastidious Strains

1. Open the vial according to enclosed instructions. 
2. Initiate the lyophilized or frozen culture according to the instructions described in the previous chapter, “Getting Started with an ATCC Bacterial Strain”. Be sure to rehydrate the entire pellet for optimal recovery. 
3. Aseptically transfer this aliquot back into the broth tube. Mix well. 
4. Use several drops of the suspension to inoculate the recommended agar slant and/or plate.
5. Incubate the tubes and plate at the recommended temperature and atmospheric conditions. The incubation period will vary between species.

Propagation of Bacteriophages and Fastidious Strains

1. Bacteriophages

   ATCC bacteriophages should be propagated in their respective bacterial host strain.

   To recover phage from freeze-dried or thawed liquid nitrogen vials:

1. Prepare an actively growing broth culture of the recommended host strain before opening the phage specimen. The host should be in early log phase. 
2. Add approximately 0.25 mL of the recommended host broth to a freeze-dried phage. 
3. Pre-warm plates of the recommended medium in an incubator. Overlay the surface with 2.5 mL of melted 0.5% agar (same medium) that contains one or two drops of the freshly grown host. The soft agar should be maintained at 43°C to 45°C until ready to pour. It may be advisable to use a water bath. Allow the overlay to harden. 
4. The re-hydrated phage can be serially diluted using a 1:10 dilution scheme by passing 0.25 mL of the phage into a tube containing 2.25 mL of the broth medium for freeze-dried vials; 0.5 mL of the phage into a tube containing 4.5 mL of the broth media for frozen vials. Repeat for as many passages as desired.
5. One drop of each dilution is spotted on the surface of the prepared plates. Allow this to dry. Three to four dilutions can be place on each plate. After 24 hours incubation, lysis should be visible. At the higher dilutions, individual plaques should be countable. 

6. NOTE 3:

   Spotting the phage on plates makes visualizing the lysis easier. If phage is added directly to soft agar before pouring plates, hazy or tiny plaques may be difficult to see. Resistant host bacteria may also mask plaque formation.

   Many strains may also be titrated without a soft agar overlay. Pipette approximately 1.0 mL of the host onto the surface of each plate. After tilting plate to ensure the entire surface is covered, the excess liquid is aspirated off. After the surface dries, the various dilutions of the phage are dropped onto the surface as before. (See: NOTE 3)



To propagate phage

1. Phage may be propagated by preparing plates with the soft agar/host overlay as above and covering the surface with approximately 0.5 mL of the concentrated phage. Or, alternatively, you may add the phage directly to the melted agar/host before pouring over the plates. For larger amounts, large-size T flasks can be prepared with the recommended agar, and approximately 12.0 mL of melted soft agar/host poured over the surface. Phage is then allowed to run over hardened surface. Phage may also be added directly to melted soft agar before pouring as described above. 
2. After 24 hours incubation, or when lysis is observed, the soft agar is scraped off the surface of the agar plates. Centrifuge at about 1000 rpm for 25 minutes to sediment the cellular debris and agar. Conserve the supernatant. 

3. NOTE 4:

   Broth propagation methods may also be employed with most phage. Unless otherwise noted in the product sheet, ATCC uses the Adams agar overlay method as described in M. H. Adams’ Bacteriophages2 for routine phage production.

   This supernatant is passed through a .22 µm Millipore filter and the filtrate stored at 4°C to 8°C. Lysates should remain viable under refrigeration for long periods. They may also be frozen with or without cryoprotectant. If available, liquid nitrogen storage is the best method for long term storage. Most phage can also be freeze dried. ATCC uses double strength skim milk mixed half-and-half with the filtrate. (See: NOTE 4)

2. Bacteroidaceae, Anaerobic

   Many Bacteroidaceae species require anaerobic conditions for growth. ATCC recommends using pre-reduced media that was either freshly prepared or previously prepared and stored under anaerobic conditions.

   Media can be prepared with reducing agents and stored in anaerobic environments such as anaerobic chambers. To prepare pre-reduced media, add 0.1 mL of a reducing agent for each 5-10 mL of mixture and let it sit for a minimum of 30 minutes. Any of the following reducing agents are appropriate: 1.5% Na₂S*9H₂O, 3% cysteine, or 5% coenzyme M.

   Pre-reduced Anaerobically Sterilized (PRAS) media is also available commercially. Under anaerobic conditions, the media is boiled free of molecular oxygen, a reducing agent is added, and the media is then autoclaved and dispensed.

1. Rehydrate the vial contents under anaerobic conditions with 0.5 mL of the recommended broth medium. 
2. Aseptically transfer this aliquot to 5 mL of the same broth. At this time, you may inoculate an agar slant of the same medium and a blood agar plate, using 0.1 mL of the cell suspension for each. 
3. Incubate the broth and slants at 37°C under anaerobic conditions. Anaerobic conditions may be obtained by the methods described in the section above, “Bacterial Growth Conditions”. 
4. Incubate the blood agar plates anaerobically for colony formation or aerobically for aerobic contamination check. 
5. After an appropriate incubation period, which is strain dependent, growth should be evident by turbidity in the broth and by colonies on the anaerobic agar surfaces. An aerobic blood plate should show no growth.

3. Bdellovibrio sp.

   Members of the Bdellovibrio genus (e.g., ATCC^® No. 27047™) are host-specific predators of Gram-negative bacteria. Therefore, it is necessary to establish a growing culture of the host strain before opening the vial of the predator. Below, we describe a basic procedure on how to culture Bdellovibrio.

1. In advance, culture the appropriate host strain according to the recommended growth conditions. 
2. Rehydrate the freeze-dried pellet of Bdellovibrio sp. with 0.5 mL of the recommended medium and transfer the suspension to a sterile test tube.
3. Add approximately 0.5 mL of the growing host strain. 
4. Melt the agar in tubes containing 2.5 mL each 0.6% semi-solid agar and place the tubes in a water bath set at 45°C. 
5. Add 0.2-0.3 mL of the host/predator suspension to each tube of melted agar and pour over the surface of a pre-warmed plate of recommended agar medium.
6. Incubate plates aerobically at 30°C for 2-3 days. Observe daily until plaques or clearings are seen. 
7. Scrape off the overlay into a test tube and centrifuge at low speed to sediment the agar and cellular debris. The Bdellovibrio should be in the supernatant. Check for tiny, rapidly moving cells under a wet mount.

4. Borrelia burgdorferi

   B. burgdorferi is a fragile, sensitive organism which must have the appropriate medium and conditions for growth. The supplementation of rabbit serum to the required Barbour-Stoenner-Kelly (BSK) medium is essential for the growth of this organism. Additionally, fresh medium enhances growth, and medium older than one month should not be used.

1. Thaw and aseptically transfer the entire contents of the liquid nitrogen vial to a tube containing 5-6 mL of fresh recommended medium. Mix well. 
2. Transfer one-tenth of the cell suspension to two or three other tubes of fresh medium. 
3. Incubate the bacteria at 32°C to 37°C under microaerophilic conditions. 
4. Growth usually occurs after 48 hours, although some strains may take several days to grow. Acid formation during growth will change the medium to a light or yellowish orange color. Turbidity is not evident. Cells can be monitored under dark-field microscopy as long spiral rods with twitching motility.

5. Leptospira sp.

   Leptospira is an aerobic strain that is fairly difficult to culture. ATCC recommends culturing this bacterium in Leptospira Medium supplemented with agar (ATCC medium formulation 1470). (See: NOTE 5)

1. NOTE 5:

   The formulations for media used by ATCC can be found on the ATCC website. Please note that most microbial media are not currently sold at ATCC.

   Aseptically withdraw the cell suspension from the thawed vial and inoculate a 10 or 12 mL tube of recommended medium, inserting the pipette just below the surface of the semi-solid medium. Aliquots of 0.5 mL from this tube may be used to inoculate additional tubes if needed. An aerobic blood plate may also be streaked to test for purity. 
2. Incubate test tubes with screw-caps slightly tightened with the blood plate aerobically at 30°C.
3. Initial growth may take from 6 to 20 days depending on the strain. Observe growth as a slight tight band forming just below the surface of the medium. The band thickens as incubation continues. Cellular morphology under dark-field microscopy shows active spiral cells with flexing motility. The aerobic blood plate should show no signs of growth.

6. Campylobacter sp.

   Campylobacter species are often difficult to grow and maintain. Many Campylobacter species, such as C. concisus, C. mucosalis, and C. showae, require a Brucella albimi broth or trypticase soy agar medium that has been supplemented with formate and fumarate.

1. Rehydrate the contents of the vial with 0.5 mL of the recommended medium. Aseptically transfer the suspension to 5.0 mL of the same broth. 
2. Inoculate prepared agar plates, agar slants, or broth tubes with 0.1 mL aliquots of the suspension. Screw caps on tubes loosely to allow for gas exchange. 
3. Incubate the cultures at 37°C under microaerophilic conditions using an anaerobe jar and a Campylobacter microaerophilic gas generator with an active catalyst or any other suitable system which gives a final atmospheric mixture of 3-5% oxygen and 10% carbon dioxide. 
4. Initially, 3-7 days of incubation may be required before visible growth is evident. Later subcultures should require only 2-3 days.

7. Helicobacter sp.

   Helicobacter species have been isolated from a variety of animals, including humans. They require microaerophilic growth conditions and can be grown on trypticase soy agar supplemented with 5% defibrinated sheep blood. Below, we describe the biphasic method of Helicobacter revival. This method provides the best recovery and most rapid growth as compared to other methods of propagation.

1. Add 0.5 mL of the recommended broth medium to the vial.
2. Mix the contents with the tip of the pipette until the pellet is rehydrated. 
3. Add 0.4 mL of the suspension to a fresh agar slant of recommended medium. Add the remaining 0.1 mL to a fresh agar plate.
4. Place the plates and test tube slant cultures into an anaerobe jar with an active catalyst and a microaerophilic gas generator Gas Pak or within an incubator that can be set up for microaerophilic conditions (3-5% oxygen and 10% carbon dioxide). 
5. Incubate cultures at 37°C for 72 hours. 
6. After 72 hours of incubation, you should observe small colonies on the surface of the agar plate and slant. There should be heavy growth in the liquid portion of the biphasic slant.

8. Legionella pneumophila

   L. pneumophila strains are nutritionally fastidious and very sensitive to the caliber of the recommended growth medium, charcoal-yeast extract (CYE) Medium (buffered). ATCC recommends that the pH of the medium be checked when the temperature of the medium is cool. Additionally, the medium should be stored in the dark as exposure to light may result in the accumulation of peroxides, which may be inhibitory to bacterial growth

1. Store vials at 4°C until ready to use. 
2. Rehydrate the freeze-dried material with 0.5 mL of the recommended broth. 
3. After the pellet has dissolved, transfer the vial contents to a test tube containing 5-6 mL of broth. Aliquots of this broth may be transferred to additional tubes of broth, to agar slants, or onto agar plates for purity and viability checks. 
4. Incubate the cultures at 37°C under atmospheric conditions of 5% carbon dioxide. Strains should show growth after 48 hours.

9. Mollicutes

   Mollicutes are a class of bacteria that are distinguished by their lack of a cell wall. Rather, most members of this class contain sterols in their cell membrane to increase membrane rigidity. Some genera included in this category include Mycoplasma, Spiroplasma, and Ureaplasma. Bacteria in this class tend to require a rich growth medium and are often sensitive to overgrowth.

1. Using the recommended medium, pipette 2.0 mL of broth into a sterile tube. Prepare additional tubes, each containing 4.5 mL of the broth. 
2. Open the freeze-dried vial as directed. Dissolve the pellet using approximately 0.5 mL of the broth from the tube containing 2.0 mL to the freeze-dried pellet. withdraw the suspension and transfer it back into the tube. 
3. Perform a serial dilution by transferring 0.5 mL from the first tube to a tube with 4.5 mL, and then 0.5 mL from the second tube to a third tube, etc. This dilution series is important for titering purposes as well as to keep the culture in varying stages of growth. Many mollicutes strains die out when cultures become alkaline. 
4. Incubate the tubes under the recommended culture conditions and temperature for each strain. The incubation period will differ between strains. For strains requiring anaerobic conditions, ATCC recommends that an anaerobe jar or other appropriate procedure is used.

10. Green and Purple Sulfur Bacteria

    Green and purple sulfur bacteria are anaerobic or microaerophilic groups of bacterial strains that are capable of photosynthesis. Unlike algae, they do not use water as their reducing agent; rather, they use hydrogen sulfide.

1. Transfer the growing culture to fresh recommended medium, filling each 125 mL screw-capped bottle to the top. 
2. Incubate the culture at room temperature under incandescent light of 2,000-3,000 lux until the elemental sulfur (i.e., milkiness) that formed disappears. 
3. Feed the cultures with 1.0-2.0 mL of sterile neutral sulfide solution.

Bacterial cell counts are necessary in order to establish or monitor bacterial growth rates as well as to set up new cultures with known cell counts.⁴ Bacterial cultures can be titered via determining the number of colony forming units per milliliter (CFU/mL) or by measuring the optical density at a wavelength of 600 nm (OD₆₀₀).

A spectrophotometer is also used to titer bacterial suspensions though determining the optical density, or absorbance, of a sample. The concentration of a bacterial culture is measured by projecting a beam of light, at a single wavelength of 600 nm, through the suspension within a transparent cuvette. When the beam of light passes through the sample, some light is absorbed while remaining light is quantified by a photometer. Generally, the more concentrated a solution is, the more light will be absorbed, and the higher the OD measurement will be. To ensure that the OD₆₀₀ reading generated by the spectrophotometer is the OD of the bacterial suspension and not that of the medium, blank the machine before use with a mediumonly control.