Biochemical reactions commonly used in microbial identification !
I. Experimental Summary <br> Some bacteria have the ability to synthesize amylase, which can secrete extracellular amylase. Amylase can hydrolyze starch to maltose and glucose, and the starch will not turn blue when it is hydrolyzed.
The lipase produced by bacteria can decompose the fat in the medium to produce glycerol and fatty acids. Fatty acids can lower the pH of the medium and can be tested by adding neutral red as an indicator to the oil medium. The neutral red indication range is pH 6.8 (red) to 8.0 (yellow). When the bacteria break down fat to produce fatty acids, red spots appear in the medium surrounding the colonies.
Some bacteria secrete proteases to break down gelatin and produce small molecules. If the bacteria have the ability to break down gelatin, the medium can change from the original solid state to the liquid state.
Milk contains mainly lactose, casein and other ingredients. The use of milk by bacteria mainly refers to the decomposition and utilization of lactose and casein. Litmus is often added to the milk as an acid-base indicator and a redox indicator. The litmus is lavender when it is neutral, red when it is acidic, blue when it is alkaline, and partially or completely discolored when it is reduced. The use of bacteria for milk can be divided into three situations:
1. Acid coagulation : After the bacteria ferment lactose, a lot of acid is produced, which makes the litmus milk turn red. When the acidity is high, the milk can be solidified. This is called acid coagulation.
2. Coagulase coagulation : Some bacteria can secrete chymosin to coagulate casein in milk, which occurs in a neutral environment. Usually, the bacteria also have the ability to hydrolyze proteins, thereby producing an alkaline substance such as ammonia, which causes the litmus to turn blue.
3. Deuteration : Casein is hydrolyzed to make the milk into a clear and transparent liquid. The deuteration can be carried out under acidic conditions or under alkaline conditions, and the litmus pigment is generally reduced by fading.
Second, the main reagents <br> starch medium: beef extract peptone medium plus 0.2% soluble starch;
Grease medium: beef extract peptone medium plus peanut oil 10mL, 0.6% neutral red aqueous solution 1mL;
Gelatin liquefaction medium: peptone 5g, gelatin 100 ~ 150g, water 1000mL, pH 7.2 ~ 7.4, sterilized at 115 ° C for 20min.
Litmus milk medium: milk powder 100g, litmus 0.075g, water 1000 mL, pH 6.8, sterilized at 121 ° C for 15 min.
Third, the experimental materials
Escherichia coli (E. coli), Bacillus subtilis (Bacillus subtilis), Staphylococcus aureus (Staphylococcus aureus), Enterobacter aerogenes (Enterobacter aerogenes), Lactobacillus sticky Alcaligenes (Alcaligenes viscolactis), Pseudomonas aeruginosa (Psudomonas aeruginosa), Proteus (Proteus vularis)
Fourth, the experimental steps
Starch hydrolysis test
1) Preparation of starch medium plate: The starch medium cooled to about 50 ° C after melting is poured into a sterile plate, and solidified to form a plate.
2) Inoculation: Draw two parts at the bottom of the plate with a marker, write the name of each fungus in each part, take a small amount of the test bacteria with the inoculating loop, and connect it to the center of the corresponding part of the surface of the medium, one of which is The species should be Bacillus subtilis as a control strain.
3) Culture: The inoculated plate was placed in a 28 ° C incubator for 24 h.
4) Detection: Remove the plate, open the plate lid, add a small amount of iodine solution to the plate, and gently rotate to make the iodine solution evenly spread the entire plate. If a colorless transparent ring appears around the colony, the starch has been hydrolyzed, indicating that the bacteria has the ability to break down starch. The size of the test strip can be used to illustrate the strength of the test strain for hydrolyzing starch.
2. Oil hydrolysis test
1) Place the triangular flask containing the oil and fat medium in a boiling water bath, take it out and shake it thoroughly to distribute the oil evenly, then pour it into a sterile dish and solidify it into a flat plate.
2) Inoculation: Inoculate on both sides of the same plate, one of which is Staphylococcus aureus as a control. The cells were cultured in an incubator at 37 ° C for 24 hours. After removal, the color of the plate moss was observed. If red spots appeared, the fat was hydrolyzed, and the reaction was positive. The red spot size indicates the strength of the test strain for hydrolyzing fat.
3. Gelatin liquefaction test
1) Inoculation: Inoculate Escherichia coli or Enterobacter aerogenes in gelatin medium by puncture inoculation.
2) Culture: Incubate in a 20 ° C incubator for 48 h. If the bacteria are not long at 20 ° C, they should be cultured at the optimum temperature.
3) Observation results: Observe whether the medium is liquefied or the shape after liquefaction.
Because gelatin solidifies below 20 °C, self-liquefaction when it is higher than 25 °C, if it is cultured above 20 °C, it should be observed in ice bath when observation, if gelatin is liquefied by bacteria, even at low temperature gelatin It will not solidify any more.
4. Litmus milk test
1) Inoculation: Alcaligenes faecalis and Pseudomonas aeruginosa are inoculated into litmus milk medium.
2) Culture: The inoculated tubes were incubated at 37 ° C for 7 days, and a non-inoculated litmus milk medium was used as a control.
3) Observation of results: The culture was taken out, and the test tube which was not inoculated with any bacteria was used as a control to observe changes in the growth of different bacteria after inoculation.
Beijing Baiou Bowei Biotechnology Co., Ltd. provides micro-industry products for inquiries and purchases for enterprises, institutions, research institutes and schools at all levels! The main products of the website are: quality control strains, standard strains, more than 40,000 strains of Chinese microbial strains, more than 10,000 strains of cell lines/strains of Staphylococcus aureus, Escherichia coli (Escherichia coli), Salmonella, Aspergillus niger, Pseudomonas aeruginosa, Bacillus subtilis, Streptococcus, Candida albicans, Shigella, etc. are commonly used strains.
I. Experimental Summary <br> Some bacteria have the ability to synthesize amylase, which can secrete extracellular amylase. Amylase can hydrolyze starch to maltose and glucose, and the starch will not turn blue when it is hydrolyzed.
The lipase produced by bacteria can decompose the fat in the medium to produce glycerol and fatty acids. Fatty acids can lower the pH of the medium and can be tested by adding neutral red as an indicator to the oil medium. The neutral red indication range is pH 6.8 (red) to 8.0 (yellow). When the bacteria break down fat to produce fatty acids, red spots appear in the medium surrounding the colonies.
Some bacteria secrete proteases to break down gelatin and produce small molecules. If the bacteria have the ability to break down gelatin, the medium can change from the original solid state to the liquid state.
Milk contains mainly lactose, casein and other ingredients. The use of milk by bacteria mainly refers to the decomposition and utilization of lactose and casein. Litmus is often added to the milk as an acid-base indicator and a redox indicator. The litmus is lavender when it is neutral, red when it is acidic, blue when it is alkaline, and partially or completely discolored when it is reduced. The use of bacteria for milk can be divided into three situations:
1. Acid coagulation : After the bacteria ferment lactose, a lot of acid is produced, which makes the litmus milk turn red. When the acidity is high, the milk can be solidified. This is called acid coagulation.
2. Coagulase coagulation : Some bacteria can secrete chymosin to coagulate casein in milk, which occurs in a neutral environment. Usually, the bacteria also have the ability to hydrolyze proteins, thereby producing an alkaline substance such as ammonia, which causes the litmus to turn blue.
3. Deuteration : Casein is hydrolyzed to make the milk into a clear and transparent liquid. The deuteration can be carried out under acidic conditions or under alkaline conditions, and the litmus pigment is generally reduced by fading.
Second, the main reagents <br> starch medium: beef extract peptone medium plus 0.2% soluble starch;
Grease medium: beef extract peptone medium plus peanut oil 10mL, 0.6% neutral red aqueous solution 1mL;
Gelatin liquefaction medium: peptone 5g, gelatin 100 ~ 150g, water 1000mL, pH 7.2 ~ 7.4, sterilized at 115 ° C for 20min.
Litmus milk medium: milk powder 100g, litmus 0.075g, water 1000 mL, pH 6.8, sterilized at 121 ° C for 15 min.
Third, the experimental materials
Escherichia coli (E. coli), Bacillus subtilis (Bacillus subtilis), Staphylococcus aureus (Staphylococcus aureus), Enterobacter aerogenes (Enterobacter aerogenes), Lactobacillus sticky Alcaligenes (Alcaligenes viscolactis), Pseudomonas aeruginosa (Psudomonas aeruginosa), Proteus (Proteus vularis)
Fourth, the experimental steps
Starch hydrolysis test
1) Preparation of starch medium plate: The starch medium cooled to about 50 ° C after melting is poured into a sterile plate, and solidified to form a plate.
2) Inoculation: Draw two parts at the bottom of the plate with a marker, write the name of each fungus in each part, take a small amount of the test bacteria with the inoculating loop, and connect it to the center of the corresponding part of the surface of the medium, one of which is The species should be Bacillus subtilis as a control strain.
3) Culture: The inoculated plate was placed in a 28 ° C incubator for 24 h.
4) Detection: Remove the plate, open the plate lid, add a small amount of iodine solution to the plate, and gently rotate to make the iodine solution evenly spread the entire plate. If a colorless transparent ring appears around the colony, the starch has been hydrolyzed, indicating that the bacteria has the ability to break down starch. The size of the test strip can be used to illustrate the strength of the test strain for hydrolyzing starch.
2. Oil hydrolysis test
1) Place the triangular flask containing the oil and fat medium in a boiling water bath, take it out and shake it thoroughly to distribute the oil evenly, then pour it into a sterile dish and solidify it into a flat plate.
2) Inoculation: Inoculate on both sides of the same plate, one of which is Staphylococcus aureus as a control. The cells were cultured in an incubator at 37 ° C for 24 hours. After removal, the color of the plate moss was observed. If red spots appeared, the fat was hydrolyzed, and the reaction was positive. The red spot size indicates the strength of the test strain for hydrolyzing fat.
3. Gelatin liquefaction test
1) Inoculation: Inoculate Escherichia coli or Enterobacter aerogenes in gelatin medium by puncture inoculation.
2) Culture: Incubate in a 20 ° C incubator for 48 h. If the bacteria are not long at 20 ° C, they should be cultured at the optimum temperature.
3) Observation results: Observe whether the medium is liquefied or the shape after liquefaction.
Because gelatin solidifies below 20 °C, self-liquefaction when it is higher than 25 °C, if it is cultured above 20 °C, it should be observed in ice bath when observation, if gelatin is liquefied by bacteria, even at low temperature gelatin It will not solidify any more.
4. Litmus milk test
1) Inoculation: Alcaligenes faecalis and Pseudomonas aeruginosa are inoculated into litmus milk medium.
2) Culture: The inoculated tubes were incubated at 37 ° C for 7 days, and a non-inoculated litmus milk medium was used as a control.
3) Observation of results: The culture was taken out, and the test tube which was not inoculated with any bacteria was used as a control to observe changes in the growth of different bacteria after inoculation.
Beijing Baiou Bowei Biotechnology Co., Ltd. provides micro-industry products for inquiries and purchases for enterprises, institutions, research institutes and schools at all levels! The main products of the website are: quality control strains, standard strains, more than 40,000 strains of Chinese microbial strains, more than 10,000 strains of cell lines/strains of Staphylococcus aureus, Escherichia coli (Escherichia coli), Salmonella, Aspergillus niger, Pseudomonas aeruginosa, Bacillus subtilis, Streptococcus, Candida albicans, Shigella, etc. are commonly used strains.
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