Bio-chemical reactions commonly used in microbial identification

experimental principle
some bacteria have the ability to synthesize amylase, can secrete extracellase. Amylase can hydrolysate starch into maltose and glucose, and iodine no longer turn blue after starch hydrolyzing.
produced by bacteria can break down
and
in the culture base to produce glycelor and fatty acids. Fatty acids can
the
medium pH and can be tested by adding neutral red as an indicator to the fat medium. The neutral red indication range is pH6.8 (red) to 8.0 (yellow). When bacteria break down fat to produce fatty acids, red spots appear in the medium around the bacteria.
some bacteria secrete
proteases
break down gelatins to produce small molecular substances. If bacteria have the ability to break down gelatin, the culture base can change from the original solid state to a liquid state.
milk mainly contains lactose, casein and other ingredients. The use of bacteria in cow's milk mainly refers to the decomposition and utilization of lactose and casein. Stone is often added to cow's milk
as an
and redox indicator. The stone is lilac when neutral, red when acidic, blue when alkaline, and partially or completely decolored when reduced. The use of bacteria on cow's milk can be divided into three cases:
(1) acid coagulation: bacteria ferment lactose, the production of many acids, so that the stone cow's milk red, when the acidity is very high, can make the cow's milk solidification, this is called acid coagulation.
(2) coagulase coagulation: Certain bacteria secrete lactase, which condenses casein in cow's milk, which occurs in a neutral environment. Usually the bacteria also have the ability
hydrolysing
proteins, resulting in alkaline substances such as ammonia, which turn the stone to blue.
(3)effect: casein is hydrolysed, turning cow's milk into a clear and transparent liquid.can be carried out under acidic or alkaline conditions, and the general stone pigment is reduced and faded.experimental material
1, live material: E. coli (E. coli ( E. coli, Bacillus subtilis, Staphylococcus aureus, Enterobacter aerogenes, Alcaligenes viscolactis, Psudomonas aeruginosa, Proteus v.
2, medium: starch medium: beef paste protein medium plus 0.2% soluble starch; pH7.2 to 7.4, 115 degrees C sterilization 20min. Stone cow milk culture: milk powder 100g, stone milk 0.075g, water 1000 mL, pH6.8, 121 degrees C sterilization 15 min.
3,
reagents
: Lugo's iodine liquid.experimental
, flat dishes, vaccination rings, alcohol lamps,
test tubes
needles and so on.Experimental Method
(i) Starch hydrolysis test
1, preparation starch medium plate: after melting and cooling to about 50 degrees C starch medium poured into a sterile flat dish, to be solidified into a flat plate.
2, inoculation: with a marker pen at the bottom of the plate in two parts, in each part of the name of the bacteria, with the inoculation ring to take a small amount of bacteria to be tested, point to the center of the corresponding part of the culture base surface, one of the strains should be hasty bacillus to do control bacteria. ★ can you explain why?
3, culture: the inoculated flat dish at 28 degrees C
thermostat
culture 24h.
4, testing: take out the plate, open the lid of the flat dish, drip with a small amount of iodine on the plate, gently rotate, so that the iodine liquid evenly spread the entire plate. If a colorless transparent circle appears around the bacterium, the starch has been hydrolysed, indicating that the bacteria has the ability to break down starch. The size of the transparent circle can be used to indicate the strength of the hydrolytic starch ability of the test strain.
(ii) Grease hydrolysis test
1, the triangular bottle containing grease medium is placed in a boiling water bath melting, removed and fully oscillating, so that the grease is evenly distributed, and then poured into the sterile flat dish, to be solidified into a flat plate.
2, inoculation: inoculated on both sides of the same flat dish, one of which is Staphylococcus ausumococcus as a control bacteria. Place in a 37-degree C thermostat to culture 24h, after removal to observe the color of flat moss, if red spots appear, that is, the fat is hydrolysed, this reaction is positive. The size of the red spots indicates the strength of the test strain's hydrolytic fat capacity.
(iii) Gelatin liquefact test
1, vaccination: vaccination with puncture inoculation of E. coli or E. coli production in gelatin medium.
2, culture: put 20 degrees C thermostat in the culture 48h. If the bacteria are not long at 20 degrees C, they should be cultured at the right temperature.
3, observation results: to observe the culture of the condition of liquefaturization and the shape of liquefaturization.
in gelatin solidified at less than 20 degrees C, higher than 25 degrees C when self-liquefaifying, if the bacteria cultured at more than 20 degrees C, observation should be placed in an ice bath to observe, if gelatin is liquefy by bacteria, even at low temperature gelatin will not solidify.
(iv) Stone cow's milk test
1, vaccination: the sticky milk production of Bacillus and copper-green false monocytobacteria inoculated into the stone calf milk medium.
2, culture: the inoculated test tube at 37 degrees C constant temperature culture 7d, in addition to retaining an unvaccinated stone bovine milk culture as a control.
3, the results of observation: take out the culture, not inoculated with any bacteria test tube as a control, to observe the changes after the growth of different bacteria inoculated.