CN118064538A - New haemophilus influenzae non-blood-source identification medium - Google Patents
New haemophilus influenzae non-blood-source identification medium Download PDFInfo
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Landscapes
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Abstract
The invention provides a new haemophilus influenzae blood-free identification medium, which is prepared by adding egg white, egg yolk, hemin, xylose and neutral red into a basic medium. The culture medium of the invention does not need to be additionally added with animal blood, has simple formula, low cost and easily obtained materials, solves the problem of self-made culture medium in areas with difficult blood sources, adapts to the requirements of clinical diagnosis and treatment, can enable haemophilus influenzae to grow rapidly, can generate visible bacterial colonies after being cultured on the bacterial culture medium for 24 hours, has no difference with the commercial chocolate agar culture medium in bacterial colony number, and eliminates the possibility of carrying blood-borne pollution by animal blood. Meanwhile, haemophilus influenzae can be identified directly by colony color, and the detection time limit of related infection is greatly shortened.
Description
Technical Field
The invention belongs to the technical field of microorganism culture media, and particularly relates to a novel haemophilus influenzae blood-source-free identification culture medium.
Background
Haemophilus influenzae is an important pathogen for lower respiratory tract infection, and can also cause endocarditis, biliary tract infection, peritonitis, nephropathy, genitourinary tract infection and other diseases. The haemophilus has higher requirement on separation and culture, is easily interfered by normal flora, and is extremely easy to generate omission in the culture and detection process. Bacterial culture is a gold standard for diagnosing bacterial infectious diseases, and can rapidly and accurately culture and identify haemophilus so as to play a key role in diagnosing and treating the infectious diseases.
Haemophilus influenzae is taken as a causticizing bacterium, has a slow growth and proliferation speed, and is cultured for 24 hours for 18-24 hours to form tiny, colorless and transparent microcolonies. A more off-white colony formed after 48 hours. The haemophilus influenzae needs a nutrient-rich culture medium, the growth is strongly dependent on X, V factors, and animal blood needs to be added into the culture medium to promote the growth. Although the culture medium containing animal blood is capable of culturing haemophilus, the rate of separation of haemophilus is not ideal. Mainly because the V factor which depends on the growth of the haemophilus influenzae is an unstable substance, the V factor is easily damaged by the influence of heat treatment factors in the preparation process of the culture medium, and meanwhile, certain enzymes in blood from different sources and bactericidal antibodies in serum can damage the V factor, so that the separation culture of the haemophilus is influenced.
The conventional culture medium for separating and culturing the haemophilus influenzae is a chocolate agar culture medium which consists of main components such as casein tryptone pancreatin digestate, meat stomach digestate, heart pancreatin digestate, yeast extract powder, corn starch, sodium chloride, agar, animal blood and the like. The culture medium has complex composition, high use cost and must be added with a large amount of blood. Blood is not only costly and difficult to obtain, but also is prone to bring about blood-borne substances, thus bringing about a series of difficulties in culturing, separating, diagnosing and mass-producing the bacteria.
The total number of haemophilus influenzae and human infection is 9, wherein the most common infection strains are haemophilus influenzae, and the haemophilus influenzae is a colorless transparent non-hemolytic microcolonie on a chocolate culture medium, and the haemophilus influenzae is similar in morphology and indistinguishable. The basis of preliminary identification is to obtain a purified single colony, and conduct relevant test culture for 18-24 hours to observe results, so that the whole culture identification process takes a long time, and diagnosis and treatment support cannot be provided for clinic in time.
Based on the method, a new haemophilus influenzae non-blood origin identification medium is provided, which can enable haemophilus to grow faster, and can generate larger colonies after being cultured on the cell medium for 24 hours, and the size and the number of the colonies are not significantly different from those of commercial chocolate medium; the influence of heat and other factors on the V factor is eliminated, and the growth of haemophilus influenzae is facilitated; meanwhile, the possibility that the added blood products carry blood-borne pollution is avoided, and haemophilus influenzae can be identified directly through colony colors.
Disclosure of Invention
The invention aims to solve the technical problems of the prior art and provides a new haemophilus influenzae blood-source-free identification medium for solving the problems in the background art.
In order to solve the technical problems, the invention adopts the following technical scheme: a new haemophilus influenzae non-blood-source identification medium is prepared by adding egg white, egg yolk, hemin, xylose and neutral red into a basic medium;
Wherein the basic culture medium comprises 20g/L peptone, 4.5g/L potato extract powder, 5.5g/L sodium chloride, 14g/L agar and 12.5g/L glycerol.
As a further explanation of the invention, the egg white and the yolk are separated by aseptic operation, and are respectively mixed by glass fragments for standby.
As a further illustration of the invention, the added volume percentage concentrations of the egg white and the yolk in the culture medium are 5% and 15%, respectively.
As a further illustration of the invention, the chlorine heme is dissolved by 0.4% NaOH, the mass volume concentration of the chlorine heme is 0.03g/L, the xylose is 10g/L, and the neutral red is 0.025g/L.
A new method for using haemophilus influenzae non-blood source identification culture medium comprises the steps of completely dissolving and recovering haemophilus influenzae freeze-dried strain with brain heart infusion broth, inoculating the strain into the culture medium for culture at 37 ℃ for 24-48 hours in a carbon dioxide incubator.
Compared with the prior art, the invention has the following advantages:
1. the culture medium provided by the invention ensures that the growth state of haemophilus influenzae is good, greatly promotes the growth speed of haemophilus influenzae, and can obtain enough haemophilus influenzae in a short time.
2. The invention discovers and proves that the eggs contain the V factor for the first time, meets the growth requirement of the haemophilus influenzae on the V factor, and eliminates the influence of heat and other factors on the V factor.
3. The culture medium provided by the invention eliminates the possibility of carrying blood-borne pollution by animal blood.
4. The culture medium provided by the invention has the advantages of simple formula, low cost and easily obtained materials, solves the problem of self-made culture medium in areas with difficult blood sources, and meets the requirements of clinical diagnosis and treatment.
5. The invention can directly identify the haemophilus influenzae by colony color, and greatly shortens the time limit of the test report of related infection.
Drawings
FIG. 1 is a 24h culture of haemophilus influenzae inoculated with egg medium (without sugar and indicator added) and chocolate medium in an example of the invention;
Wherein the left graph is egg culture medium, and the right graph is chocolate culture medium;
FIG. 2 shows the coloration of colonies of Haemophilus influenzae and Haemophilus parainfluenzae on a chromogenic medium in an embodiment of the invention
Wherein the left graph shows red colonies of influenza, and the right graph shows colorless colonies of parainfluenza.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The embodiment of the invention provides a technical scheme that: a new haemophilus influenzae non-blood-source identification medium is prepared by adding egg white, egg yolk, hemin, xylose and neutral red into a basic medium;
Wherein the basic culture medium comprises 20g/L peptone, 4.5g/L potato extract powder, 5.5g/L sodium chloride, 14g/L agar and 12.5g/L glycerol.
The egg white and the egg yolk are separated by aseptic operation, and are respectively mixed uniformly by glass fragments for standby.
The added volume percentage concentrations of the egg white and the egg yolk in the culture medium are 5% and 15%, respectively.
The chlorine heme is dissolved by 0.4% NaOH, the mass and volume concentration of the chlorine heme is 0.03g/L, the xylose is 10g/L, and the neutral red is 0.025g/L.
When preparing culture medium, firstly purchasing fresh egg, soaking in 75% alcohol for 30min, sterilizing egg surface with 1% iodophor, sterilizing egg white and egg yolk separated by aseptic operation, respectively placing into autoclaved yellow wide-mouth bottle with glass fragments, and fully shaking and uniformly mixing for use.
Weighing the following raw materials: 20g of peptone, 4.5g of potato soaked powder, 5.5g of sodium chloride, 14g of agar, 12.5g/L of glycerol, 0.03g of hemin, 10% NaOH solution, 10g of xylose and 0.025g of neutral red, dissolving the raw materials by using ultrapure water, fixing the volume to 800mL, and regulating the pH value to 7.2-7.6;
sterilizing at 121deg.C under high pressure for 20min, cooling to 50-60deg.C, adding 50mL egg white and 150mL egg yolk into the culture medium, mixing, pouring into a 90mm sterile plate, cooling, and preserving at 2-6deg.C.
Experimental example the preparation of the comparative haemophilus influenzae conventional medium was as follows:
S1, weighing the following raw materials: 10g of casein peptone pancreatin digestate, 5g of meat stomach digestate, 3g of cardiopancreatin digestate, 5g of yeast extract powder, 1g of corn starch, 5g of sodium chloride and 12g of agar;
s2, dissolving the raw materials by using ultrapure water, fixing the volume to 1000mL, and regulating the PH value to 7.2-7.6;
s3, autoclaving at 121 ℃ for 20min;
S4, after the temperature of the culture medium is reduced to 80-85 ℃, adding 100mL of aseptic defibrinated sheep blood, fully and uniformly mixing, pouring the mixture into a sterile plate with the thickness of 90mm, cooling, and preserving at the temperature of 2-6 ℃.
Growth condition comparison:
Resuscitates Haemophilus influenzae, subcultures for 48 hours by using a commercialized chocolate culture medium, takes purified bacterial colonies to regulate the bacterial liquid concentration to 0.5 McN, 1.5X10 7 bacteria count/mL, adopts a double-ratio dilution method, takes 100ul of bacterial suspension with 0.5 McN, adds 900ul of diluent, sequentially dilutes the bacterial liquid concentration to 10 7,106,105,104, shakes and mixes uniformly, and each 10ul of liquid is absorbed and inoculated to an egg culture medium and a chocolate culture medium respectively, and the commercialized chocolate culture medium is repeated for 3 times. The bacterial growth and colony morphology of each plate were observed according to the bacterial growth cycle.
As can be seen from FIG. 1, H.influenzae forms larger colonies in the egg medium for 24h, which are not different in size from the commercial chocolate medium.
Table 1: colony count comparison of haemophilus influenzae quantitatively inoculated in egg medium and chocolate medium (10 4 CFU/mL)
| Colony count | Test 1 | Test 2 | Test 3 | Average value of | Colony count |
| Egg culture medium | 78 Pieces | 80 Pieces of | 71 Pieces of | 76 | 7600CFU/mL |
| Chocolate culture medium | 65 Pieces of | 72 Pieces of | 75 | 71 Pieces of | 7100CFU/mL |
From the results in Table 1, it can be seen that there was no difference between the number of bacteria in the egg medium and the number in the chocolate medium in the quantitative culture of H.influenzae 10 4. The egg culture medium is very favorable for separating and culturing the haemophilus influenzae.
And (3) comparing the strain preservation efficiency:
The haemophilus influenzae is placed on an egg culture medium and a chocolate culture medium for 48 hours of pure culture, and is preserved in a refrigerator at the temperature of 2-6 ℃ and transferred to a commercial chocolate culture medium respectively at 1d, 3d, 5d, 7d, 15d, 30d, 60d and 90d, and the survival rate condition of the haemophilus influenzae in different preservation periods is compared and observed.
Table 2: egg culture medium and chocolate culture medium strain preservation efficiency comparison
As can be seen from Table 2, the Haemophilus influenzae on the egg medium survived more than 60d in refrigerator preservation, and the Haemophilus influenzae on the chocolate medium survived 30d in refrigerator preservation, and the 60d was not survived. The egg culture medium is more beneficial to the short-term preservation of the haemophilus influenzae.
Comparing the identification effect;
the haemophilus influenzae and haemophilus parainfluenzae are respectively inoculated on an egg culture medium and a chocolate culture medium, and are cultured for 24-48 hours in a carbon dioxide incubator at 37 ℃. The color of the colonies on the culture was observed separately.
As can be seen from FIG. 2, the haemophilus influenzae and the haemophilus parainfluenza respectively show red and colorless colonies on the egg culture medium, and the haemophilus influenzae and the haemophilus parainfluenza are colorless and transparent colonies on the chocolate culture medium, so that the egg culture medium can accurately identify the haemophilus influenzae.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (5)
1. A new haemophilus influenzae non-blood-source identification medium is characterized in that: the culture medium is prepared by adding egg white, egg yolk, hemin, xylose and neutral red into a basic culture medium;
Wherein the basic culture medium comprises 20g/L peptone, 4.5g/L potato extract powder, 5.5g/L sodium chloride, 14g/L agar and 12.5g/L glycerol.
2. The novel haemophilus influenzae non-blood-source identification medium of claim 1, wherein the egg white and the yolk are separated by aseptic technique, and are mixed with glass fragments for use.
3. The novel haemophilus influenzae non-blood-borne identification medium of claim 1, wherein the egg white and yolk are added to the medium at a volume percentage concentration of 5% and 15%, respectively.
4. The novel haemophilus influenzae non-blood-source identification medium of claim 1 wherein the chlorine-containing heme is dissolved in 0.4% naoh at a concentration of 0.03g/L, xylose 10g/L, neutral red 0.025g/L.
5. A method for using a newly prepared haemophilus influenzae non-blood source identification culture medium is characterized in that after the haemophilus influenzae freeze-dried strain is completely dissolved and revived by brain heart-containing immersion liquid broth, the haemophilus influenzae freeze-dried strain is inoculated into the culture medium of claim 1 for culture at a temperature of 37 ℃ for 24-48 hours in a carbon dioxide incubator.
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