Biochemical Tests of Bacteria (also known as biochemical reactions) refer to the process of determining metabolic products through biochemical methods, where different bacteria possess distinct enzyme systems, leading to varied abilities to decompose substrates and thus produce different metabolic products. Biochemical tests of bacteria mainly include carbohydrate biochemical tests, amino acid and protein metabolism tests, carbon and nitrogen source utilization tests, enzyme metabolism tests, etc. Different bacteria vary in their protein-decomposing capabilities: generally, extracellular enzymes first break down proteins into short peptides or amino acids, which then enter bacterial cells and are further decomposed into amino acids by intracellular enzymes.
Aladdin's gelatin liquefaction test reagent, also called gelatin liquefaction medium, is mostly used for gelatin liquefaction experiments. Its principle is that gelatin liquefaction medium itself solidifies below 20°C and liquefies spontaneously above 25°C. When inoculated with bacteria that secrete gelatinase, the gelatin is hydrolyzed into small-molecule substances, destroying its colloidal state. After cultivation, even when the temperature is below 20°C, the gelatin in the medium no longer solidifies and changes from the original solid state to a liquid state, which is defined as gelatin liquefaction. Based on this, it can be judged whether the bacteria have the ability to decompose proteins. This ability varies significantly among different bacterial species, so it has long been used as a characteristic for identifying and determining bacteria. For example, bacteria of the genera Staphylococcus, Proteus, and Bacillus all have liquefaction ability, while Escherichia coli does not. Gelatin liquefaction medium is mainly composed of high-purity gelatin, peptone, deionized water, etc. After sterile processing, it is mostly a pale yellow viscous liquid or jelly at room temperature. If a liquid form is required, it should be heated to dissolve before use. This reagent is for research use only and is not suitable for clinical diagnosis or other purposes.
Self-prepared Materials:
1. Test bacteria: Escherichia coli, Enterobacter aerogenes, Bacillus subtilis, etc.
2. Inoculating loops, alcohol lamps, glass test tubes or petri dishes, constant temperature incubators.
Operating Procedures (For Reference Only):
1. Slightly heat the gelatin liquefaction medium and dispense it into glass test tubes or petri dishes.
2. Take the young bacterial strains cultured for 18–24 hours and inoculate them into glass test tubes or petri dishes via stab inoculation. Set up an uninoculated control group. Incubate the cultures in a constant temperature incubator at 20°C for 2–5 days. For bacteria that do not grow well at 20°C, the incubation temperature can be adjusted to 36°C.
3. Observe the bacterial growth, medium liquefaction status, and liquefaction morphology every day. If the gelatin itself liquefies due to high incubation temperature, place the inoculated cultures in a refrigerator at 2–8°C for approximately 15–30 minutes without shaking. Take them out immediately after cooling and observe the results. Persistent liquefaction indicates a positive test result. If the experiment is performed in petri dishes, drop the reagent onto the colonies spot-inoculated on the medium plates. A positive result is indicated by a clear zone forming around the colonies within 10–20 minutes; otherwise, the result is negative.
The common morphologies of gelatin liquefaction are shown in the figure below:
Precautions:
1. Ensure aseptic technique during operation to avoid microbial contamination.
2. The reagent is mostly a pale yellow viscous liquid or jelly at room temperature. If a liquid form is required, heat it to dissolve before use.
3. For bacteria that cannot grow at 20°C, incubation must be carried out at their optimal required temperature. When observing the results, remove the test tubes from the incubator and place them in an ice bath to cool for 15–30 minutes before checking the degree of liquefaction.
4. For your safety and health, wear a lab coat and disposable gloves during operation.
5. Please use the reagent as soon as possible after opening to prevent any impact on subsequent experimental results.
