Bacterial Culture Chongqing Scope

Types of culture media:

Culture media are artificial products of various nutrients required for bacterial growth and reproduction. Suitable culture media can make bacteria grow and reproduce rapidly in vitro, making it easier to separate and identify bacteria. It can be divided into basic culture media, nutrient culture media, selective culture media, identification culture media, anaerobic culture media and special culture media.

01# Basic culture media

Contains only the most basic nutrients required for bacterial growth, is the most widely used, and is the basis for preparing a variety of culture media. Common ones include broth culture media and agar culture media.

02# Nutrient culture media

Adding organic substances such as glucose, blood, serum, ascites or yeast extract to the basic culture media can meet the growth needs of bacteria with higher nutritional requirements or for bacterial enrichment. For example, eggs, potatoes, glycerol, etc. are added to the culture media of Mycobacterium tuberculosis.

03# Selective culture media

A culture medium made by taking advantage of the different sensitivities of different types of bacteria to chemical substances, which allows isolated bacteria to multiply in large quantities and inhibits the growth of other bacteria. The inhibitors contained in the culture medium can inhibit the growth of non-target bacteria or make them grow poorly, which is conducive to the detection and identification of target bacteria. The selected culture medium is mostly solid plates, which are used to separate certain specific bacteria from specimens.

04# Identification culture medium

Certain specific components, such as sugars, alcohols and indicators, are added to the culture medium to check various biochemical reactions of bacteria for the purpose of identifying and identifying bacteria.

05# Culture medium for anaerobic bacteria

Obligatory anaerobic bacteria must grow under anaerobic conditions, so it is necessary to add reducing agents such as cysteine and sodium thioglycolate to the culture medium to reduce the redox potential in the culture medium, and it should be isolated from the outside air so that the culture medium itself is an anaerobic environment.

06# Special culture medium

It is used to culture some bacteria that need to grow under special conditions. Such as hypertonic salt enrichment culture medium, hypertonic sugar enrichment culture medium, modified Kagan's culture medium, etc.

Preparation of culture medium:

The main process of preparing general culture medium is basically similar, including mixing, dissolving, adjusting pH, filtering, packaging, sterilization, testing and preservation.

Laboratory precautions:

Bacterial culture must be operated at any time to prevent contamination and the spread of pathogens, that is, aseptic operation. The precautions of the bacterial laboratory are as follows:

01#

Bacterial culture should be carried out in an inoculation hood or sterile room. Laboratories with conditions can be carried out in a clean bench.

02#

When using an inoculation loop to separate and transplant bacteria, sterilization is required before and after use. Flame sterilization is generally used.

03#

When taking specimens or transplanting from culture bottles or test tube cultures, they must be passed over the flame 2 to 3 times before opening the bottle mouth, tube mouth or closing it. Do not allow bacterial materials to contaminate countertops and other objects.

04#

If the test tube is accidentally broken and the bacterial solution is contaminated, do not panic, report it immediately to the person in charge, and then treat the contaminated countertop or ground with 3% lysol or 5% carbolic acid, and soak for at least 30 minutes.

05#

After finishing the work, irradiate with UV light for 30 minutes, or wipe the countertop with 3% Lysol and wash your hands.

Inoculating loops and needles:

In microbiology, inoculation is the process of adding bacteria to a culture medium so that they can multiply there. Vaccines, serum, or other antigenic substances are often introduced into the body in order to increase immunity to a certain disease.

Inoculating loops and needles are handheld, compact instruments that are used to introduce microorganisms such as bacteria or yeast into a plate or tube of growth medium prior to incubation, multiplication, or growth. The inoculum is usually transported and spread into a liquid culture medium, streaked onto or pierced into a solid agar medium, or both.

The inoculation loop and inoculation needle consist of three parts, namely the loop (needle) part, the metal handle part and the insulation handle part. The inoculation loop and inoculation needle usually use electric heating (nickel) wire. The diameter of the loop varies with the purpose of use, generally 2-4mm, and the length of the loop and needle is 40-50mm.

The inoculation loop (needle) should be sterilized before and after use. Put the end of the inoculation loop (needle) upright in the flame, burn the nickel wire part red, and then rotate the metal handle of the inoculation loop (needle) through the flame 3 times for sterilization. After cooling, use it to take bacteria or test specimens.

Immediately after using the inoculation loop (needle), heat the contaminated nickel wire part in the reducing flame (inner flame) to dry the bacteria or specimens attached to the end of the loop (needle) to prevent the residual bacteria or specimens on the loop (needle) from being suddenly exposed to high heat and splashing, thereby polluting the environment and causing infection risks. Then move it to the oxidizing flame (outer flame) to burn it red for sterilization, and finally pass the metal handle part back and forth in the flame 3 times. After use, the inoculation loop (needle) should be placed on the rack immediately and never discarded to avoid burning the table or other objects.

Inoculation operation area

In order to avoid environmental pollution and airborne bacteria contaminating the culture during the inoculation process, inoculation should be carried out in an inoculation hood or sterile room. In addition, the necessary equipment for bacteriological laboratories also includes sterile workbenches, biosafety cabinets and biosafety laboratories.

01# Inoculation hood

There are many styles of inoculation hoods, which can be made of wooden frames and glass, or made of plexiglass. Before use, the inoculation hood should be wiped lightly with 3% carbolic acid or Lysol. It should be equipped with an ultraviolet sterilization lamp, which can be irradiated before use to ensure that there is no dust and bacteria in the hood. After the operation is completed, the interior should be cleaned immediately and the hood should be disinfected.

02# Sterile workbench

Also known as super clean bench, it currently adopts vertical laminar airflow. The air filtered by the pre-filter in the negative pressure box is pressed into the static pressure box by a variable speed centrifugal fan, and then filtered by a high-efficiency filter for secondary filtration. The clean airflow blown out from the air outlet surface passes through the working area at a certain and uniform cross-sectional wind speed, taking away dust particles and microbial particles, thus forming a dust-free and sterile working environment. When in use, the UV sterilizer should be turned on 50 minutes in advance, and turned off and the blower started after 30 minutes. It is strictly forbidden to store unnecessary objects in the purification area to keep the clean airflow pattern undisturbed.

03# Aseptic room

A sterile room, also known as a clean room, is a small room installed inside the laboratory for aseptic operations. There should be air filtration devices, UV sterilizers, etc. in the room.

04# Biosafety cabinet

Currently, biosafety cabinets are mostly used in microbiological experiments. They are negative pressure filter exhaust cabinets designed to protect the operator, the laboratory environment, and the experimental materials when operating infectious experimental materials such as primary cultures, bacterial (viral) strains, and diagnostic specimens, so as to avoid exposure to infectious aerosols and splashes that may be generated during the above operations.

05# Biosafety laboratory

Biosafety laboratory (Biosafety Laboratory), referred to as "BSL laboratory". It refers to a laboratory built through standardized experimental design, configuration of experimental equipment, and use of personal protective equipment. Structurally, it consists of a primary protection barrier (safety equipment) and a secondary protection barrier (facilities). Different combinations of safety equipment and facilities for laboratory biosafety protection constitute four levels of biosafety protection, with level one being the lowest.