Methods of resuscitation and activation of common strains

First, formulate a plan

When we obtain a strain of bacteria, before resuscitation activation, we must first understand the growth characteristics of the strain, determine the nutritional needs of the strain, growth temperature, gas environment, osmotic pressure, pH and salinity and other conditions, select the appropriate medium and culture environment, and formulate a recovery activation program. Strains usually purchased from the Culture Preservation Center are accompanied by instructions, and you can follow the instructions. If there is no instruction manual, you can check the literature (recommended to check the Berger Bacteriological Manual or common bacteria identification manual) to confirm the growth conditions of the strain, and then formulate a plan.

2. Strain treatment and inoculation operation

After obtaining the strain, in principle, activation and preservation should be carried out as soon as possible. If it cannot be activated immediately, it should be stored in the corresponding environment according to the instructions.

(1) If the preserved strain is in the form of lyophilized powder or the form of carrier preservation, a small amount of normal saline or non-selective broth medium can be added to dissolve and suspend, inoculated to the plate by scribing or coating, or aspirated the bacterial liquid into liquid medium for culture.

(2) If the preserved strain is glycerol cryopreservation or liquid preservation form, it can be scribbled or coated to the plate after rapid melting at room temperature, or directly added to the liquid medium for culture.

(3) If the preserved strain is in the form of porcelain beads, a single porcelain bead can be picked and rolled on the plate several times or directly added to the liquid medium for incubation.

(4) If the preserved strain is in beveled or semi-solid form, the moss can be directly picked and transferred to a plate or liquid medium for culture.

Third, the choice of culture medium

1. The composition of the medium used to activate the strain usually includes the following components:

(1) Basic nutritional components: such as peptone, beef soaking powder, yeast soaking powder, etc., mainly providing carbon sources, nitrogen sources and trace elements;

(2) Sodium chloride: maintain a certain osmotic pressure environment;

(3) Phosphate: maintain the relative stability of pH;

(4) Special ingredients: such as serum, cysteine, heme chloride, etc., to promote the growth of specific bacteria. According to the growth characteristics of the strain, select the appropriate medium or add ingredients to the specific medium to improve to meet the growth needs of the strain.

2. There are the following types of commonly used activation media:

(1) Pancreatic soy uxin liquid/agar medium, nutritious broth/agar medium, brain heart immersion broth/agar medium: rich in nutrients, suitable for the recovery activation culture of most common bacteria.

Note: You can add blood, serum, vitamins, heme chloride and other components to the culture medium to increase nutrition and cultivate some nutrient-demanding bacteria.

(2) Blood plate: 5%-10% of defibrous sheep blood or rabbit blood can be added using blood agar base, and 5%-10% of defibrous sheep blood or rabbit blood can be added to the medium such as TSA or BHI as the basis to make blood plates, which can cultivate some bacteria with high nutritional requirements, such as Campylobacter, Haemophilus, Neisseria, Clostridium, Streptococcus, Helicobacter, etc.

(3) Chocolate plate: Different from the blood plate, the use of light heating to make the red blood cell split in the blood to explain the release of various internal growth factors, the plate made is chocolate-colored, the medium is often used for the culture of Haemophilus, Neisseria and Streptococcus.

(4) MRS broth/agar: pH of partial acidity and high sugar content, suitable for the growth of lactic acid bacteria, often used for the culture of lactic acid bacteria such as lactobacillus, lactococci, bifidobacteria and so on.

(5) 2216E liquid/agar medium: similar to the composition and osmotic pressure of seawater, commonly used for the culture of marine bacteria, such as Vibrio parahaemolyticus, Vibrio algalolyticus, Vibrio hashib, etc., growing vigorously in medium containing about 3% salinity.

(6) Herpetic meat culture medium, thioglylate fluid medium: commonly used for the culture of anaerobic Clostridium, such as Clostridium gas-producing Clostridium, Clostridium spore, etc.

Note: When using liquid medium to cultivate anaerobic bacteria, cover the surface of the medium with liquid paraffin wax to isolate the air, without putting into an anaerobic incubator, you can directly cultivate anaerobic bacteria (thioglymate fluid medium can naturally form an anaerobic environment, no need to cover liquid paraffin wax, nor do you need to anaerobic culture).

(7) BCYE: dedicated to the culture of Legionella.

(8) Sagrade glucose liquid/agar medium: The higher sugar content is conducive to the growth of fungi, and is often used to cultivate fungi such as mold and yeast.

(9) Mycoplasma broth, Arginine Mycoplasma broth: serum needs to be added for the culture of Mycoplasma pneumoniae, Oral Mycoplasma, etc.

(10) ISP2, Kolmo-1 medium: commonly used for the culture of actinomycetes.

(11) GAM medium: The medium is highly nutritious and can be used for the culture of a variety of aerobic anaerobic bacteria, such as Bacteroides, Clostridium, Bifidobacterium, etc.

(12) Roche medium, Middle Brook 7H10: for the culture of mycobacteria.

Note: When activating strains, it is generally not advisable to use selective media with bacteriostatic effects, such as TSA/TSB for Staphylococcus aureus activation, but it is generally not appropriate to use selective plates such as mannitol sodium chloride agar and Baird-parker agar for activation.

Fourth, the cultivation conditions

(1) Temperature: The most suitable growth temperature for most bacteria is 30-37 ° C, the most suitable growth temperature for fungi and actinomycetes is 20-30 ° C, the most suitable growth temperature for Campylobacter is 42 ° C, the most suitable growth temperature for thermophilic bacteria is about 55 ° C or higher, and the cold bacteria usually grow below 20 ° C.

(2) Gas environment

Obligate aerobic type: such as Pseudomonas aeruginosa, Bacillus subtilis, etc., require oxygen to grow, and can be cultivated in normal air.

Facultative anaerobic type: such as E. coli, Salmonella, etc., aerobic and anaerobic can grow well, in normal air can be cultivated.

Obligate anaerobic type: such as Bifidobacterium, Clostridium, Bacteroides, etc., oxygen will have a toxic effect on these bacteria, inhibit growth, so it is necessary to cultivate in a dedicated anaerobic incubator or anaerobic tank. Most anaerobic bacteria require 5% carbon dioxide to promote growth.

Micro-aerobic type: such as Campylobacter, Helicobacter, etc., suitable for growing in an environment of 5%-10% oxygen, and need to be cultured in a microaerobic incubator or culture tank.

Carbon dioxide: The initial culture or isolation of some bacteria requires a carbon dioxide environment to promote growth, such as Streptococcus pneumoniae, Legionella, etc.

(3) Other conditions: such as photosynthetic bacteria need light.

V. Recommended activation culture method for common bacteria

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Strain name

Activation of culture methods

Escherichia coli, Salmonella, Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Shigella, Proteus, Enterobacter sakazakia, Listeria, Klebsiella, Saretella, Enterococcus, Aeromonas aeromonas, Yersinia enterocolitis, BurkeHoldella onion, etc

TSA/TSB or NA/NB, incubate at 30-36 °C for 18-24 h

Candida albicans, Saccharomyces cerevisiae, etc

SDA/SDB/PDA, 20-25 °C for 3-5 days

Aspergillus niger

SDA/PDA, culture at 20-25 °C for 5-7 days or culture to produce black spores

Streptomyces albicans

CULTURE ISP2 medium 28-30 °C for 3-5 days

Clostridium aeronasculata, Clostridium spore, Clostridium difficile

Thioglyceate fluid medium or herpes medium was cultured at 30-36 °C for 24-48 h, or anaerobic culture for blood plates and Colombian plates at 30-36 °C for 24-48 h.

Note: Clostridium viability in thioglycolate fluid medium is shorter and can be stored for a longer time using herpes meat medium.

Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus terrestris infantis bifidobacterium, Streptococcus thermophilus, etc

MRS agar/broth 36± 1 °C for 24-48 h (Bifidobacterium bifidobacterium requires anaerobic culture)

Vibrio parahaemolyticus, Vibrio traumatis, Vibrio cholerae, Vibrio algae, etc

2216E liquid/agar medium or 3% sodium chloride TSA, culture at 30-36 °C for 18-24 h

Streptococcus pneumoniae, hemolytic streptococcus

Blood plates were cultured at 36±1 °C for 24-48 h

Garcinia cambogia micrococcus

TSA or nutrient agar is cultured at 30-36 °C for 24-48 h

Campylobacter jejuni, Campylobacter colonia, etc

Blood plates are micro-aerobic cultured at 42 °C for 24-48 h

Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae, etc

Blood plate or chocolate plate, 36±1 °C culture for 24-72 h, the initial isolation of Neisseria usually requires 5% -10% carbon dioxide environment

Bacteroides fragile

Anaerobic culture of hemophane, GAM, or BBE36 ± 1 °C for 24-48 h

Mycoplasma oral cavity, Mycoplasma pneumoniae

Arginine Mycoplasma broth (oral) or Mycoplasma broth (pneumonia) 36±1 °C culture for 3-7 days, the first resurrection is usually more difficult to cultivate, sometimes takes several weeks, the middle also needs to be blindly transmitted twice, in the environment containing 5% -10% carbon dioxide is easier to culture

Legionella Bozemania, Legionella pneumophila, etc

BCYE36 ± 1 °C for 3-7 days, the initial culture grows better in a 5% carbon dioxide environment

Strain Resuscitation Operation Video: https://www.ixigua.com/6862566424200937987

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