General microbiological identification technology.

First, morphological structure and culture characteristics observation 1, microorganism morphological structure observation is mainly through dyeing, under the microscope of its shape, size, arrangement, cell structure (including cell walls, cell membranes, nuclei, whiplash, spores, etc.) and dyeing characteristics to observe, intuitively understand the characteristics of bacteria in morphological structure, according to the different morphological structure of different microorganisms to achieve differences, identification of microorganisms.
2, the cell population formed by bacterial cells on the surface of solid cultures is called colony.
different microorganisms grow and reproduce in a certain medium, the formation of fall features are very different, while the same bacteria under certain conditions, culture characteristics have a certain stability.
can be used to distinguish between microorganisms.
, the observation of microbial culture characteristics is also an important part of microbial testing and identification.
1) The culture characteristics of bacteria include the following: on solid medium, observe the size, shape, color (pigment is water-soluble or fat-soluble), gloss, transparency, texture, bulge shape, edge characteristics and migration.
surface growth (bacterial membrane, ring) turbidity and precipitation in liquid culture.
semi-solid culture base puncture vaccination to observe the movement and diffusion.
(Figure 3-8)1. Dot 2. Round 3. Silk 4. Irregular 5. Fake Root 6. Spindle 7. Flat 8. Bump 9. Bump 10. Pad 11. Umbilical 12 . The edges are neat 13. Wave 14. Cracked 15. Mesh 16. Silky 17. Curl 18. Silky 19. Spike 20. Beaded 21. Dredging 22. Root 23. Fake root 24.silky 25.beaded 26.nipple-like 27.fluffy 28.tree root-shaped 29.measured cup-shaped 30.radish-shaped 31.funnel-shaped 32.cystic 33.. Layered 34. floc-like 35. ring-shaped 36.membrane 37.membrane-like 2) the culture characteristics of mold yeast: Most yeasts do not have silky bodies, the formation of bacteria on solid media and bacteria are very similar, but bigger and thicker than bacterial flora.
liquid culture is similar to bacteria, with uniform growth, precipitation, or formation of a membrane on the liquid surface.
mold has branched silky body, mycelium thick length, in the condition of suitable media, mycelium infinite elongation along the surface of the media spread.
mold of the base of mycelium, gas and spores are often with different colors, so the edge and center of the bacteria, front and back colors are often different, such as penicillin: spore turquoise, gas mycelium colorless, sterilization brown.
mold forms floc-like, fluffy and cobweb-like floc-like floc-like, cobweb-like floc-like floc-like on solid culture surfaces.
, physiological and biochemical test Microbial biochemical reaction refers to the use of chemical reactions to determine the metabolites of microorganisms, biochemical reactions are often used to identify some of the form and other aspects of the microorganisms are not easy to distinguish.
microbial bio-chemical reaction is one of the important basis in the classification and identification of microorganisms.
bio-chemical reactions commonly used in microbiological testing are: 1, sugar enzyme testIng Different microorganisms have very different ability to break down and utilize sugars, or can or cannot be used, can be used, or gas production or non-gas production.
can be tested with indicators and fermentation tubes.
test method: sterile operation, with inoculation needles or rings to remove a small amount of pure culture, inoculated in fermented liquid culture tube, if semi-solid culture, then with vaccination needles for puncture vaccination.
after vaccination, put 36 to 1.0 degrees C culture, daily observation results, check the color of the culture base has changed (acid production), small inverter has no bubbles, small bubbles are also positive for gas production, if it is a semi-solid culture, then check along the puncture line and pipe wall and pipe bottom there are small bubbles, and sometimes can see that the inoculation bacteria have no power, if there is power, culture can be dispersed growth.
test is mainly to check the bacteria on a variety of sugars, alcohols and glycosides fermentation capacity, so as to identify a variety of bacteria, so each test, often need to be inoculated multiple tubes at the same time.
commonly used indicators are phenolic red, bromophenol purple, bromine thyme and An-drade indicators.
2, starch hydrolyzing test Some bacteria can produce enzymes that break down starch and hydrolysate starch into maltose or glucose.
starch hydrolysing, iodine no longer turns blue.
test method: with 18 to 24h pure culture, coated in starch agar slope or plate (a plate can be partitioned inoculation, test several cultures) or directly transferred to starch broth, cultured at 36 to 1 degree C 24 to 48h, or cultured at 20 degrees for 5 days.
the iodine reagent is then directly immersed in the culture surface, and if it is a liquid culture, a few drops of iodine reagent are added to the test tube.
immediately, the positive test (starch is broken down) is that the agar culture is dark blue, there is no colorless transparent ring around the culture, or the color of the broth is unchanged.
negative reaction is no transparent ring or the broth is dark blue.
process of the starch hydrolysis system, so the test results are related to the ability of the bacteria to produce amylase, the culture time, the amount of starch and pH in the culture base, etc.
medium pH must be neutral or slightly acidic to be most appropriate for pH7.2.
starch agar plate should not be stored in the refrigerator, so it is prepared in time.
3, V-P test Some bacteria in glucosewater medium can break down glucose to produce acetone acid, acetone acid shrink, de-pyridine into acetyl methyl methanol, the latter in the strong alkali environment, oxidized by oxygen in the air to diacetyl, diacetyl and proteinin the production of red compounds, called V-P (plus) reaction.
test method: 1) O'Meara method: inoculation of the experimental bacteria in a universal culture base, culture 48h at 36 x 1 degree C, culture 1 ml plus O'Meara reagents (plus 0.3% creatine creatine or creatine creatine or Creatinine creatinine 40% sodium hydroxide solution) 1 ml, shaking test tube 1 to 2min, quietly placed at room temperature or 36 to 1 degree C thermostatic box, if 4h does not show Ir red, that is, judged negative.
also advocates in the 48 to 50 degrees C water bath placed 2h after determining the outcome.
2) Barritt's method: inoculate the experimental bacteria in a universal culture base, culture them for 4 days at 36 x 1 degree C, and add 5 degrees C alpha-phenol (2-na-phthol) pure alcohol solution 0.6 ml in 2.5 ml of culture. Add 40% potassium hydroxide solution 0.2 ml, shake 2 to 5min, positive bacteria often immediately appear red, if no red appears, static placed at room temperature or 36 to 1 degree C thermostatic box, such as 2h still does not appear red, can be judged negative.
3) Fast method: 0.5% crea acid solution 2 drops placed in a small test tube, pick acid-producing reaction of the triglyceride agar slope culture a vaccination ring, emulsified inoculation, add 5% alpha-phenol 3 drops, 40% sodium hydroxide solution 2 drops, after vibration placed 5min, the results were determined.
cultures that do not produce acid cannot be used.
test is generally used for the identification of various strains of E. coli.
When used in other bacteria such as Bacillus aureus and staphylococcus, phosphates in general culture can hinder the production of acetyl methyl alcohol and should be omitted or replaced with sodium chloride.
4, methyl red (Methyl Red) test E. coli genus can ferment glucose, in the process of decomposition of glucose to produce acetone acid, further decomposition, due to different ways of sugar metabolism, can produce lactic acid, serum acid, acetic acid and formic acid and a large number of acid products, can reduce the medium pH to pH4.5 below, so that methyl red indicator red.
test method: pick a small number of new pure culture to be tested, inoculated in a universal culture base, cultured at 36 x 1 degree C or 30 degrees C (better at 30 degrees C) 3 to 5 days, from the next day, daily culture 1 ml, plus methyl red indicator 1 to 2 drops, positive bright red, weak positive light red, negative yellow.
results can be determined if they are found to be positive or as low as the 5th day.
is an acidic indicator with a pH range of 4.4 to 6.0 and a pK value of 5.0.
is below pH5.0, with acidity and enhance yellow, above pH5.0, then with alkalinity and enhance yellow, in pH5.0 or close up and down, may not change color obviously enough, at this time should be extended culture time, repeat the test.
5, Imdole test some bacteria can break down tryptophan in protein , the production of radon.
presence of radon can be demonstrated by a color-reflecting reaction.
paracetamol and metformin to form rose pyridine, a red compound.
test method: the small amount of pure culture to be tested in the test media tube, at 36 to 1C culture 24h, take about 2 ml of culture fluid, add Kovacs reagent 2 to 3 drops, light shake test tube, red For positive, or first add a small amount of ether or xylene, shake the test tube to extract and concentrate the indigo substitin, until it floats on the surface of the culture solution, and then slowly along the test tube wall to add Kovacs reagent drops, in the contact surface is red, that is, positive.
Experiments have shown that indigo-based reagents can be positive with 17 non-indigo-based compounds, if extracted with xylene or ether, etc., and then reagented, only the indigo or 5-methyl-indigo substate appears red in the solvent, so the results are more reliable.
6, nitrate reduction test Some bacteria have the ability to reduce nitrates, nitrates can be reduced to nitrites, ammonia or nitrogen.
nitrites can be tested with nitric acid reagents.
test method: before the test reagent A (sulfonamide ice acetic acid solution) and B (alpha-amine ethanol solution) test fluid 0.2 ml each equivalent mix, take the mixture reagent about 0.1 ml, added to the liquid culture or agar slope culture surface, immediately or within 10min red is positive for the test, if no red appears is negative.
test with alpha-amine, the positive red recedes quickly, so the results should be determined immediately after addition.
must have an unvaccinated culture tube as a negative control when conducting the test.
alpha-amine is carcinogenic and should be used with care.
7, Gelatin liquefact test Some bacteria have gelatinase (also known as protein hydrolyzed enzyme), gelatin hydrolyzed into peptides, and further hydrolyzed into amino acids, loss of gel properties and liquefact.
test method: pick 18 to 24h bacteria culture to be tested, with a large number of puncture inoculated in gelatin high-rise about 2/3 depth or point planted on the plate culture base.
cultured for 7 to 14 days at 20 to 22 degrees C.
high-rises can also be cultured at 36 to 1 degree C.
Daily observation results, if due to high culture temperature and make gelatin itself liquefax should not shake, sit in the refrigerator until it solidified, and then observe whether it is liquefied by bacteria, if it is indeed liquefied, is positive for testing.
the results of the plate test were observed as drip-added reagents on the drop-in reagents of the strains of the plate-point species of the culture base, and if positive, 10 to 20min should be surrounded by clear band rings.
would otherwise be negative.
8, ureaase (Urease) test some bacteria can produce urea enzymes, urea break down, produce 2 molecules of ammonia, so that the culture base become alkaline, phenolic red pink.
is not an induced enzyme, because bacteria can synthesize it regardless of the presence of substrate urea.
its activity is the most suitable pH of 7.0.
test method: pick 18 to 24h to test bacteria culture in large quantities inoculated in liquid medium tube, shake the average, culture 10, 60 and 120min at 36 to 1 degree C, respectively, observed the results.
or coated and punctured on agar slopes, do not reach the bottom, leaving the bottom for a chromic contrast.
, 2, 4 and 24h were observed, respectively, if negative should continue to be cultivated for 4 days, for final determination, to pink as positive.
9. Oxidase test Oxidase, also known as cytochrome oxidase, is the end-of-life respiratory enzyme of the cytochrome respiratory enzyme system, oxidase first oxidizes cytochrome C, and then this oxidizing cytochrome C oxidizes against benzodiacytes to produce a color reaction.
test method: 1 to 2 drops of reagents on agar slope culture or blood agar flat bacteria, positive Kovacs reagents were pink to dark purple, Ewing's improved reagents were blue.
negatives have no color change.
results should be determined within minutes.
10, Hydrogen sulfide (H2S) test Some bacteria can break down the medium containing sulfur amino acids or sulfur compounds, and the production of hydrogen sulfide gas, hydrogen sulfide with lead salts or low iron salts can produce black sediment.
test method: In the medium containing sodium sulfate and other indicators, punctured along the pipe wall, cultured 24 to 28h at 36 to 1 degree C, and the medium was black-positive.
should continue to be cultivated for 6 days.
Can also be used lead acetate paper method: the bacteria to be tested in the general nutritional broth, and then the lead acetate paper hanging over the culture base, so that it will not be splashed wet as a moderate;
the note turned black to positive.
11, triglyceride iron (TSI) agar test method: to inoculate needles to pick out suspected bacteria or pure culture, puncture inoculation and coating on the slope, placed 36 to 1 degree C culture 18 to 24h, observation results.
test can observe both lactose and sucrose fermentation acid or acid production gas (yellowing);
Glucose is decomposed to produce acid can make the slope yellow first, but because of the small amount, the small amount of acid produced, oxidized by contact with air, coupled with bacteria using nitrogen-containing substances in the medium, to produce alkaline products, so that the slope later turned red, the bottom because it is in an anaerobic state, the acid is not oxidized, so it remains yellow.
12, hydrogen sulfide-indigo substitin-power (SIM) agar test method: to inoculate needle pick bacteria or pure nutrient puncture vaccination about 1/2 depth, put 36 to 1 degree C culture 18 to 24h, observation results. br.