I. Introduction
1. Biological characteristics
Vibrio cholerae is a gram-negative bacterium that is arc-shaped or comma-shaped, sporangial, hairy, and some strains have capsulates, with a thick, long flagella at one end, and are active. The swill water-like feces of cholera patients were taken for observation of live bacteria suspension drops, and it was seen that the bacteria were extremely active, shuttling or meteor-like movements. Liquid culture drops are stained for microscopic examination, and arrangements such as "fish swarm" Vibrio can be seen. After artificial cultivation, it is easy to lose the arc shape and become rod-shaped.
The nutritional requirements are not high, grow well on ordinary medium, can grow in the pH range of 6.8-10.2, alkali resistance, grow well in alkaline peptone water or plate at pH 8.8-9.0, because other bacteria are not easy to grow at this pH, so alkaline peptone water can be used as a medium for selective proliferation of Vibrio cholerae.
Colonies on alkaline plates are 2 mm in diameter, round, smooth, transparent. Vibrio cholerae is sensitive to heat, dryness, daylight, chemical disinfectants and acids, and is resistant to low temperatures: damp heat 55 ° C, 15 minutes, 100 ° C, 1-2 minutes, add 0.5 ppm chlorine to water for 15 minutes to be killed; 0.1% potassium permanganate soaked vegetables and fruits can achieve disinfection purposes; survive only 4 minutes in normal stomach acid. Vibrio cholerae is an aerobic or facultative anaerobic bacterium that can grow at 16-44 °C at an optimal growth temperature of 37 °C.
2. Epidemiological characteristics
Vibrio cholerae is the pathogen of cholera, which is an acute and intense intestinal infectious disease, mainly manifested as severe vomiting, diarrhea, dehydration, acute onset, strong contagiousness, high mortality, and is an international quarantine infectious disease. There are 139 serogroups, of which the O1 and O139 groups can cause cholera. Vibrio cholerae group O1 and O139 group are pathogens of cholerae in the severe intestinal infectious disease cholera. In nature, humans are susceptible to Vibrio cholerae. People cause cholera by ingesting food or water contaminated with Vibrio cholerae. Vibrio cholerae entering the intestine passes through the mucus layer on the surface of the intestinal mucosa through flagellar motion, approaches the epithelial cells of the intestinal mucosa, and colonizes by the action of ordinary hairs. The environment in the intestine is conducive to the reproduction of Vibrio cholerae, which produces cholerae enterotoxin, leading to the occurrence of disease.
The main causative factor of Vibrio cholerae is cholera toxin (CT), CT is the most powerful toxin known toxin, which can cause a large amount of intestinal fluid secretion, resulting in severe vomiting and diarrhea, causing a large loss of water and dielectrics in the body, forming a severe watery diarrhea syndrome. The main lesions caused by Vibrio cholerae are caused by severe dehydration, and fingerprints are wrinkled and dried up in the subcutaneous tissue and muscles. The heart, liver, spleen and other organs are all reduced. The visceral serous membrane is dull. The intestinal lumen is highly dilated, the intestine is filled with a swill-like liquid, and the intestinal mucosa is relaxed, but the mucosal epithelium is intact and there is no ulcer. The gallbladder is filled with viscous bile. Telangular dilation of the glomeruli and interstitium, swelling, degeneration and necrosis of the tubules. Other organs also have changes such as bleeding and degeneration.
Second, the inspection process
The test reference standard is "SN/T 1022-2010 Vibrio cholerae test method in import and export food". This standard specifies the test method of Vibrio cholerae in imported and exported food, which is suitable for the test of Vibrio cholerae in food, and the test of Vibrio cholerae in environmental samples of animal feed and other food production and processing areas can be used by reference.
The Vibrio cholerae test procedure is shown in Figure 1:
Figure 1
1. Identification of culture medium and judgment of results
(1) TCBS medium
Principle: Peptone and yeast infiltration powder provide carbon and nitrogen sources, vitamins and growth factors; sodium chloride can stimulate the growth of Vibrio; sucrose is a fermentable sugar; sodium bovine bile, bovine bile powder, sodium thiosulfate and sodium citrate and a higher pH can inhibit gram-positive bacteria and coliform bacteria; sodium thiosulfate reacts with iron citrate as an indicator for detecting hydrogen sulfide production; bromothorb phenol blue and thymol blue are pH indicators; agar is a coagulant of culture media.
How to use: Weigh 89g of this product, boil and dissolve in 1000ml of distilled water, when cold to 45-50 ° C, pour into a sterile flat dish, set aside. Note: Autoclaving is not required.
Results: Vibrio cholerae was rounded on TCBS, the surface was smooth, yellow, and the diameter was 2mm to 3mm, and the specific test results were as shown in Figure 2:
Fig. 2 Growth results of Vibrio cholerae non-01 TCBS medium
(2) Gentamicin agar medium
Principle: Peptone and beef dip powder provide nitrogen sources, vitamins, minerals and growth factors; sucrose provides carbon sources; sodium chloride is used to meet the salt-loving growth requirements of Vibrio cholerae, while forming a high osmolality that is not conducive to the growth of other bacteria; sodium sulfite can stimulate the growth of Vibrio vibrio; gentamicin and potassium tellurinate and a high pH value can inhibit the growth of Gram-positive bacteria and some gram-negative heterogeneous bacteria; sodium citrate can make Vibrio cholerae immune to the influence of other inhibitory substances; Vibrio cholerae is more sensitive to acidic environments. Therefore, this pH value promotes its growth; agar is the coagulant of the medium.
How to use: Weigh 56.0g of this product, heat and stir to dissolve in 1000ml of distilled water, when cold to about 50 ° C, add sterile 1% potassium tellurium oxide solution of 0.5ml, mix well, pour into sterile dishes, set aside. No autoclaving is required.
Results: Vibrio cholerae was slightly bluish gray, translucent, flattened and slightly raised on gentamicin agar medium, such as prolonged culture or room temperature placement, and the center of the colony formed a ferrous metal tellurium precipitate, and the specific test results were shown in Figure 3:
Colonies are slightly bluish grey, translucent, flattened and slightly raised
After prolonging the incubation time, a melanomerite tellurium precipitate is formed in the center of the colony
(3) Sodium chloride trisaccharide iron agar
Principle: peptone, beef dip powder, yeast dip powder to provide carbon and nitrogen sources, vitamins and minerals; lactose, glucose, sucrose for fermentable sugars, which are decomposed by phenol red acid base indicator when the acid is decomposed, acid is yellow, alkaline is red; because some bacteria can decompose sulfur-containing amino acids, the formation of hydrogen sulfide, hydrogen sulfide and iron salts in the medium react to form a black ferrous sulfide precipitation; higher content of sodium chloride can promote the growth of Vibrio vibrio; agar is the coagulant of the culture medium.
How to use: Weigh 69.5g of this product, add 1000ml of distilled water, heat and boil to completely dissolve, aliquot test tubes, autoclave at 121 °C for 15 minutes, set aside.
Results: The reaction of Vibrio cholera on the slope of sodium trisaccharide is yellow on the bottom layer, yellow on the slope, does not produce hydrogen sulfide, does not produce gas, and the specific test results are as shown in Figure 4:
(4) Sodium chloride nutritional agar
Principle: Peptone and beef dip provide the carbon and nitrogen sources, vitamins and growth factors needed for microbial growth; high content of sodium chloride can promote the growth of Vibrio cholerae; agar is a coagulant of culture medium.
How to use: Weigh this product 36.0g, heat and dissolve in 1000ml distilled water, aliquot triangular bottle or test tube, autoclave at 121 °C for 15 min, set aside.
Results: After the colonies of Vibrio cholerae were purified and cultured on sodium chloride nutrient agar medium, the colonies appeared round, the edges were translucent and centrally opaque, and the diameter was about 3 mm.
2. Biochemical identification and result judgment
(1) Gram staining
Reagent used: Gram staining solution kit.
Principle: Through crystal violet initial dyeing and iodine liquid mordant, in the cell wall formed insoluble crystal violet and iodine complex, gram-positive bacteria due to its thick cell wall, peptidoglycan network more layers and crosslinking dense, in the case of ethanol or acetone decolorization treatment, due to water loss but make the mesh shrink, plus it does not contain lipids, therefore, can be crystalline violet and iodine complex firmly left in the wall, so that it is still purple; and gram-negative bacteria because of its thin cell wall, high outer membrane layer lipid content, peptidoglycan layer thin and poor crosslinking, After encountering decolorizers, the outer membrane dominated by lipids dissolves rapidly, and the thin and loose peptidoglycan network cannot block the dissolution of crystal violet and iodine complexes, so it is still colorless after ethanol decolorization, and then restained by red dyes such as sand yellow, which makes gram-negative bacteria red.
Results: Vibrio cholerae is a gram-negative bacterium that is arc-shaped or curved, and the staining results are as shown in Figure 6:
(2) Oxidase test
Reagent used: Oxidase test strip.
Principle: Oxidase, also known as cytochrome oxidase, is the terminal respiratory enzyme of the cytochrome respiratory enzyme system, oxidase first oxidizes cytochrome c, and then this oxidizing cytochrome C then oxidizes p-phenylenediamine to produce a color reaction. Pick individual colonies with a thin glass rod or disposable inoculation needle and apply to test strips. Turning blue or bluish-purple within 30s is positive and discoloration is negative.
Results: A single colony of pure culture on a vegetative agar was selected for oxidase test, and Vibrio cholerae was positive for oxidase, and the test results were as shown in Figure 7:
(3) Biochemical identification strip test
Reagent used: Qingdao Haibo HBI Vibrio cholerae biochemical identification strip (SN).
Reagent Introduction: Biochemical Identification for Vibrio Cholerae. Composition: ONPG, urease, gelatin, arginine bihydrase broth, ornithine decarboxylase broth, lysine decarboxylase broth, amino acid decarboxylase control, unsalted pancreatic anhydrin water, 2% sodium chloride pancreatic uchi water, 6% sodium chloride pancreatic water, 8% sodium chloride pancreatic fluorine water, 10% sodium chloride pancreatic uricone water, indigo matrix, 1% sodium chloride lactose, 1% sodium chloride fibrodiose, 1% sodium chloride arabinolate, 1% sodium chloride sucrose, 1% sodium chloride mannose, 42 ° C growth test. (SN Standard).
Operation process: take a sterile saline test tube containing 2ml, use the inoculation needle to pick part of the colonies from the sodium chloride nutrient agar plate to sterile saline, carefully grind to make a uniform bacterial suspension of 0.5 myrtellois turbidity, take a biochemical identification strip without pollution, add 100ul bacteria suspension per hole, of which the solid and semi-solid biochemical tubes are inoculated with piercing, and the ornithine, arginine, lysine and amino acid controls need to be covered with sterilized paraffin wax. After inoculation, make the label, close the lid, put into the bottom tray, and place the culture at 36 °C ± 1 °C (42 °C growth and 42 °C culture). After the culture is over, record the results of the experiment. The specific test results are shown in Figure 8:
Vibrio cholera non-01 biochemical identification result table
<col>
Biochemical projects
The result is determined
Biochemical shapes
ONPG
＋
Yellow
8% sodium chloride peptone water
﹣
Does not grow
Urease
10% sodium chloride peptone water
gelatin
liquid
1% sodium chloride mannitol
red
ornithine
purple
1% sodium chloride lactose
Does not change color
lysine
1% sodium chloride fibro disaccharide
Amino acid control
1% sodium chloride arabinose
arginine
1% sodium chloride sucrose
No salt peptone water
The growth becomes cloudy
Indigo matrix
Red ring
2% sodium chloride peptone water
42 °C growth test
6% sodium chloride peptone water
oxidase
Note: + indicates positive; - indicates negative
Precautions: 1. During the indigo matrix test, 2-4 drops of Kovacs indigo matrix reagent should be added after the end of the culture, and the observation results were observed within 4 hours after mixing; 2. Ornithine, arginine, lysine and amino acid controls should be covered with sterilized liquid paraffin.
3. Summary of results
Vibrio cholerae is rounded on TCBS, the surface is smooth, yellow, and the diameter is 2 mm to 3 mm; on gentamicin agar medium, it is slightly bluish gray, translucent, flat and slightly raised, such as prolonged culture or room temperature placement, the colony center forms a ferrous metal tellurium precipitate; the reaction on the inclined surface of sodium chloride trisaccharide iron is bottom yellow, bevel yellow, no hydrogen sulfide is produced, no gas production; the colony is round after purification and culture on sodium chloride nutrient agar medium, and the edge is translucent and centrally opaque, with a diameter of about 3 mm Identified as Gram-negative by gram staining test, arc-shaped or curved; purple on oxidase test strips, positive experimental results; growth in salt-free peptone water and 2% sodium chloride peptone water; 6% sodium chloride peptone water, 8% sodium chloride peptone water growth is positive and 10% sodium chloride peptone water does not grow; ONPG is positive; urease is negative; gelatin is positive; ornithine, lysine decarboxylase is positive; amino acid control and arginine are negative; sucrose and mannitol are positive Lactose, fibrobiose and arabinose were negative, indigo matrix test was positive, and grown in a 42°C incubator. In addition to the above tests, the detection of Vibrio cholerae can also be used for serological typing and other related tests.