Temperature cultivation mode
One of the main parameters for bacterial classification is their temperature growth optimum. Depending on which temperature is most favorable for metabolism and division, microorganisms are divided into three main groups:
- Mesophiles. The optimal temperature for their cultivation is in the range of $20–40\ ^\circ\text{C}$. This is the most significant group in medical microbiology, as the temperature optimum coincides with human body temperature. The overwhelming majority of pathogenic bacteria are mesophiles.
- Thermophiles. Heat-loving microorganisms that actively multiply at high temperatures — from $40$ to $60\ ^\circ\text{C}$. Typical representatives of thermophiles include actinomycetes, as well as some species of spore-forming bacilli.
- Psychrophiles. Cold-loving bacteria with a temperature optimum ranging from $0$ to $20\ ^\circ\text{C}$.
Methods for creating anaerobic conditions
To grow oxygen-sensitive bacteria, it is necessary to create special gas conditions. There are two main approaches to creating anaerobiosis:
- Chemical method. Based on the use of special substances that absorb oxygen from the environment (e.g., alkaline pyrogallol). In practice, sealed containers equipped with systems such as gas packs are also widely used. The main purpose of chemical methods is the cultivation of aerotolerant bacteria capable of surviving in the presence of residual oxygen.
- Mechanical (physical) method. Consists of the complete physical removal of air from the working volume followed by its replacement with an inert gas. This process requires specialized equipment, such as anaerobic jars or sealed anaerobic workstations.
Incubation parameters: time and light
In addition to temperature and gas composition, time and light regimes are crucial factors.
Time factor directly depends on the division rate (generation time) of a specific microorganism species. For most bacteria, the standard cultivation period ranges from 18 to 48 hours. However, there are slow-growing species whose diagnosis takes significantly longer:
- The causative agent of pertussis requires incubation for 5 days.
- Mycobacterium tuberculosis (the causative agent of tuberculosis) forms visible growth only after 3–4 weeks.
Light regime is vital for phototrophic microorganisms. In medical microbiology, light has important diagnostic significance when working with opportunistic mycobacteria: illumination directly affects their ability to form pigment, which is a key criterion in intraspecific identification.
Specific methods and industrial cultivation
Cultivating obligate intracellular parasites presents a particular challenge in microbiology. This group includes representatives of the genera Rickettsia, Ehrlichia, Coxiella, and Chlamydia. Since they cannot reproduce on acellular artificial media, living systems are used for their cultivation:
- Cell cultures;
- Organisms of susceptible animals or arthropods;
- Chicken embryos.
Note: Chicken embryos are suitable for cultivating most intracellular agents, as well as bacteria with a high degree of heterotrophy (e.g., Borrelia and Legionella). An exception is Ehrlichia, which do not grow on chicken embryos.
Industrial cultivation has completely different scales and goals. It is aimed at obtaining a huge microbial biomass, which is then used for the production of antibiotics, vaccines, probiotic preparations, and diagnostic agents. This process is carried out in specialized apparatuses — fermenters — and requires the strictest automatic control of all growth parameters of the bacterial population.
Briefly: answers to topical questions
Which pathogenic microorganisms are classified as psychrophiles?
Pathogenic psychrophiles and psychrotrophic bacteria include the causative agents of yersiniosis and plague.
- The causative agent of intestinal yersiniosis (Yersinia enterocolitica) has a temperature optimum of 22–28 °C and is capable of multiplying at refrigerator temperatures (4 °C).
- The causative agent of pseudotuberculosis (Yersinia pseudotuberculosis) also actively multiplies at 4 °C.
- The causative agent of plague (Yersinia pestis) has a growth temperature range of 0–40 °C with an optimum at 25 °C.
What methods are used to create anaerobic conditions during cultivation?
Physical, chemical, and biological methods of establishing anaerobiosis are used for cultivating strict anaerobes.
- Physical method — air removal with inert gas replacement in anaerobic jars and workstations, inoculation in a "deep stab" culture medium, as well as medium regeneration (boiling with rapid cooling) overlaid with liquid petrolatum or paraffin.
- Chemical method — use of gas generator sachets (such as "GasPaks"), oxygen scavengers (alkaline pyrogallol), and the addition of reducing agents to the medium (thioglycolic acid, sodium thioglycolate, cysteine).
- Biological method — Fortner's method, which consists of co-culturing with aerobes that consume oxygen.
What special nutrient media are used for growing anaerobes?
Special media and inoculation conditions are used for growing anaerobic bacteria:
- Fluid thioglycollate medium (Kitt-Tarozzi medium) — an enrichment medium for anaerobes; growth in liquid media such as Kitt-Tarozzi is accompanied by turbidity, gas production, and a specific odor.
- Thioglycollate medium — used for anaerobes; reducing agents such as thioglycolic acid, sodium thioglycolate, or cysteine can be added to the media.
- Blood agar — a solid medium for obtaining pure cultures of anaerobes; growth may be accompanied by a zone of hemolysis.
- Liver agar — polymorphic star-shaped colonies are described on it.
- Sugar agar stab — R-forms described as "fluff balls" with a dense center and lenticular S-forms.
An acronym for obligate intracellular bacteria is CERC: Chlamydia, Ehrlichia, Rickettsia, Coxiella. They require "living systems" exclusively for growth.
Class and exam questions
Why is the incubator set to 37 °C for the majority of human pathogens?
The overwhelming majority of disease-causing bacteria in humans are mesophiles. Their temperature optimum is 20–40 °C, which ideally matches the human body temperature.
How are bacteria grown if an anaerobic jar is not available?
The chemical method can be used: place cultures in a sealed container along with oxygen scavengers (alkaline pyrogallol or specialized "GasPak" envelopes). This is especially effective for aerotolerant microorganisms.
Can Chlamydia be grown on a standard nutrient medium?
No, Chlamydia (along with Rickettsia, Coxiella, and Ehrlichia) are obligate intracellular parasites. Their reproduction requires living cells: tissue cultures, chick embryos, or laboratory animals.
Why is light required for the cultivation of mycobacteria?
Light is used as a differential-diagnostic test for opportunistic mycobacteria: upon exposure to light, they begin to produce pigment, which helps to accurately identify the species.
What else to study on this topic
- Mechanisms of chemical oxygen absorption by pyrogallol
- Features of heterotrophic nutrition in Borrelia and Legionella
- Operating principles of industrial fermenters
- Why Ehrlichia are incapable of multiplying in chick embryos
- Pigment production in opportunistic mycobacteria
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