CN1224061A - Method for preparing human red blood cell culture medium - Google Patents
Method for preparing human red blood cell culture medium Download PDFInfo
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- CN1224061A CN1224061A CN 98110037 CN98110037A CN1224061A CN 1224061 A CN1224061 A CN 1224061A CN 98110037 CN98110037 CN 98110037 CN 98110037 A CN98110037 A CN 98110037A CN 1224061 A CN1224061 A CN 1224061A
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Abstract
A culture medium for human red cells features that after sterilized for 30 min, the hydrolytic casein agar is cooled to 45-55 deg.C and then mixed with human red cell liquid in a volume ratio of (5-20) : 1. Its advantages are high separation speed of bacteria and high selectivity of sensitive medicine.
Description
The present invention relates to a kind of preparation method who is used for the bacterium isolation medium, especially a kind of preparation method who is used for the isolating human red blood cell culture medium of bacterium.
At present, be used for the isolating substratum of clinical bacteria and adopt sheep blood meida and traditional separation method more, these methods are the isolating needs of incompatibility clinical bacteria, and its velocity of separation is too slow, and the information that obtains is often because of out-of-date and ineffective.Do not reach the effect that clinical desirable pathogenic agent sharp separation is identified and sensitive medicaments is selected, and conventional sheep blood source is not enough, adopts and carry the slurry back and desire the depleted red corpuscle, reach that responsive method is selected and the purpose of utilization of waste material the substituting of sheep blood.
The objective of the invention is to overcome the deficiency of above-mentioned prior art and provide a kind of bacterium to separate and the preparation method of the human red blood cell culture medium fast and accurately of drug sensitivity test.
Purpose of the present invention can be 5-20 to 45-55 ℃ with HRBC by volume by 15 pounds of 30 minutes sterilising treatment postcooling of agar that following measure reaches caseinhydrolysate: 1 mixes.
Purpose of the present invention can also reach by following measure, and caseinhydrolysate agar is by 4-24% ox meat extract powder, and 38-58% caseinhydrolysate, the starch of 1-15%, the agar of 14-34% mix the hydrolysis of back adding distilled water and form.The amount that adds distilled water is ox meat extract powder, caseinhydrolysate, starch, four kinds of component gross weights of agar 20-28 a times.Transferring PH=7.4-7.6. HRBC liquid during hydrolysis is by in red corpuscle ten sterile flask behind people's venous blood extraction blood plasma, with the stroke-physiological saline solution washing, leaves standstill, and abandoning supernatant forms.Can obtain red blood cells of type A liquid, Type B red corpuscle liquid, O type red corpuscle liquid, AB type red corpuscle liquid and each balanced mix suspension of four kinds of erythrocytes of ABO blood group system with this method.
Compared with the prior art the present invention has following advantage: 1. the human red blood cell culture medium with the preparation of this method has substituted the sheep blood meida, has solved the conventional sheep blood insufficient problem of originating.2. shortened the bacterium sepn process greatly,, be condensed to 10-18 hour, 3. improved the accuracy that sensitive medicaments is selected by original 18-72 hour.
Be that human red blood cell culture medium and sheep blood meida effect compare below
Table (1) .32 example acinetobacter haemolyticus causes infection, and secretory product is directly used in A, B, O, AB, 8 kinds of allosome substratum of ABO 16h, the speed of growth and sheep blood 24h appraisal of growth rate
| The substratum time point | A B O AB ABO sheep blood plate |
| Bacterium colony mean diameter (mm) is estimated | 2.45 the good good general well-grown of 2.52 2.60 2.47 2.44 2.59 well-growns |
Table (2), from clinical hemolytic bacteria, zone of hemolysis contrast on the Autoerythrocyte substratum
| Substratum | Zone of hemolysis width (mm) streptococcus aureus beta streptococcus pneumococcus alpha streptococcus influenzae |
| 5% red blood cell culture medium, 10% red blood cell culture medium, 20% red blood cell culture medium sheep blood meida | ????0.44?????????1.68???????0.36????????0.22???????0.32 ????0.46?????????1.70???????0.39????????0.23???????0.34 ????0.43?????????1.66???????0.35????????0.21???????0.31 ????0.45?????????1.70???????0.39????????0.23???????0.33 |
Table (3). select each 30 strain of haemolysis bacterial strain, direct separation and Culture 16h, meter is calculated in each plate 1/4 zone, and hemolytic bacteria generation zone of hemolysis width and colony diameter are relatively
| Various bacterium culture mediums | Zone of hemolysis width (mm) staphylococcal pneumonia diplococcus beta streptococcus alpha streptococcus gonorrhoea attitude plucked instrument Salmonella |
| AB AB0 sheep blood plate | 0.45 ( 1.14 ) 0.36 ( 1.12 ) 1.69 ( 0.57 ) 0.21 ( 0.43 ) 0.42 ( 1.45 ) 0.46 ( 1.13 ) 0.39 ( 1.14 ) 1.68 ( 0.57 ) 0.20 ( 0.45 ) 0.43 ( 1.47 ) 0.48 ( 1.14 ) 0.40 ( 1.13 ) 1.70 ( 0.58 ) 0.22 ( 0.48 ) 0.45 ( 1.51 ) 0.47 ( 1.14 ) 0.38 ( 1.14 ) 1.69 ( 0.58 ) 0.20 ( 0.44 ) 0.42 ( 1.47 ) 0.46 ( 1.14 ) 0.38 ( 1.15 ) 1.67 ( 0.67 ) 0.21 ( 0.44 ) 0.44 ( 1.49 ) 0.45 ( 1.14 ) 0.39 ( 1.15 ) 1.70 ( 0.58 ) 0.23 ( 0.49 ) 0.45 ( 1.51 ) ** |
Table (4), choice criteria bacterial strain extent of dilution is 10-5, is separating 16h on the allosome red blood cell culture medium fully, measures bacterium colony mean diameter and sheep blood plate and compares.
| The standard bacterium culture medium | ATCC ATCC ATCC mean diameter is estimated 25,923 25,922 27853 mm |
| Red blood cells of type A culture medium Type B red blood cell culture medium 0 type red blood cell culture medium AB type red blood cell culture medium ABO four type red blood cell equivalent are mixed platform culture medium sheep blood meida | 1.04 1.32 3.25 1.87 well-growns, 1.05 1.33 3.30 1.92 well-growns, 1.05 1.34 3.32 1.93 well-growns, 1.038 1.31 3.17 1.906 well-growns, 1.045 1.31 3.29 1.915 well-growns, 1.046 1.325 3.30 1.937 well-growns |
Table (5). the pathogenic bacterium streptococcus aureus separates the speed of growth at various human blood plate components, and colony diameter and sheep blood plate are relatively
| The standard bacterium culture medium | Each extent of dilution bacterium colony mean number (strain) 10 -1?10 -310 -510 -7Bacterium colony mean diameter (mm) is estimated |
| Red blood cells of type A culture medium Type B red blood cell culture medium O type red blood cell culture medium AB type red blood cell culture medium AB0 four type red blood cell equivalent are mixed platform culture medium sheep blood meida | ++ 150 146 100 1.042 well-growns ++ 153 142 97 1.041 well-growns ++ 152 147 96 1.042 lead well long+149 143 101 1.040 well-growns+150 145 98 1.040 well-growns ++ 153 145 99 1.041 well-growns |
Table (6). reference culture streptococcus aureus (ATCC 25923) is at the antibacterial ring of the antibacterial ring of 5%, 10% human blood plate 16h susceptibility sheep blood plate 24h susceptibility
| Substratum antibacterials (us) | Inhibition zone diameter (mm) 5% red blood cell culture medium (16h) 10% red blood cell culture medium (16h) sheep blood plate contrast (24h) |
| Penicillin G, (10) benzene azoles green grass or young crops, (75) erythromycin, (15) lincomycin, (2) pioneer 2, (15) tsiklomitsin, (30) amikacin, (30) | ????24.2????????????????24.5????????????????24.4 ????25.0????????????????25.7????????????????25.4 ????23.6????????????????25.4????????????????25.3 ????25.2????????????????26.6????????????????26.3 ????25.9????????????????29.3????????????????29.5 ????20.1????????????????20.5????????????????21.2 ????24.3????????????????25.0????????????????24.7 |
Table (7), 136 examples, smear G-diplococcus positive patient, direct susceptibility of secretory product sample and purifying 10
-3The bacterium drug sensitivity tests compares, and both reach more than 95% recombination rate.
| Substratum | The antibacterial ring coincidence rate of susceptibility sense (diameter mm) (diameter coffee) (%) behind the responsive antibacterial ring purifying of direct susceptibility |
| Allosome red blood cells of type A culture medium (25) allosome Type B red blood cell culture medium (26) allosome 0 type red blood cell culture medium (30) allosome gizzard type red blood cell culture medium (30) allosome ABO four type red blood cell mixed in equal amounts culture medium (26) sheep blood meidas (30) | ????23.5??????????????25.2??????????93.3 ????24.2??????????????24.6??????????98.4 ????24.7??????????????23.6??????????96.5 ????23.6??????????????24.0??????????98.3 ????24.1??????????????24.7??????????97.5 ????24.5??????????????24.9??????????98.3 |
Describe the present invention below in detail.
Gather people's venous blood, slowly move in the sterile flask with aseptic formality, to remain red corpuscle behind the aseptic formality extraction blood plasma, with stroke-physiological saline solution washing 2-3 time, adopt the gimmick of rotation gently during each the washing, shake 20-30 time to same direction, leave standstill the back and supernatant liquor is discarded, promptly get HRBC liquid with aseptic straw.
Getting weight ratio then is 4-24% ox meat extract powder, the 38-58% caseinhydrolysate, the starch of 1-15%, the agar of 14-34% mixes, adding people's weight then is the 20-28 distilled water accent PH=7.4-7.6 doubly of ox meat extract powder, caseinhydrolysate, starch, agar gross weight, hydrolyzed solution, 30 minutes sterilising treatment under 15 pounds of conditions and caseinhydrolysate agar.
Get the sterilising treatment postcooling to 45-55 ℃ of caseinhydrolysate agar and HRBC liquid, its volume ratio is 5-20: 1 mixes, and promptly gets to be used for the isolating human red blood cell culture medium of bacterium.
Embodiment 1, gather patient self venous blood 10ml, slowly move in the sterile flask with aseptic formality, with stroke-physiological saline solution washing 2-3 time, to adopt the gimmick of rotation gently during each the washing, shake 20-30 time to same direction, leave standstill the back and supernatant liquor is discarded, promptly get HRBC liquid with aseptic straw.
Get ox meat extract powder 5g, caseinhydrolysate 17.5g, starch 1.5g, agar 12.5g, add 1000ml distilled water, transfer PH=7.4, the caseinhydrolysate agar of 15 pounds of 30 minutes sterilising treatment, getting, sterilising treatment is chilled to 40 ℃ caseinhydrolysate agar 100ml, add the HRBC liquid 20ml that the people prepares, shake up and promptly get human red blood cell culture medium, the aseptic plate of the system of inclining, generally the plate by the 9cm diameter adds the 25ml nutrient solution.
Embodiment 2. get deposit in the 20ml blood bank 3-7 days aseptic carry slurry after, isolate aseptic red corpuscle, wash 2-3 time with stroke-physiological saline solution under the room temperature, to adopt the gimmick of rotation gently during each the washing, shake 20-30 time to same direction, firmly not excessive, in order to avoid haemolysis, leave standstill the back and supernatant liquor is discarded, promptly get HRBC liquid with aseptic straw.
Get ox meat extract powder 4g, caseinhydrolysate 38g, starch 1g, agar 14g, add 1600ml distilled water, transfer PH=7.6,15 pounds of 30 minutes sterilising treatment, get caseinhydrolysate agar, get that sterilising treatment is chilled to 55 ℃ caseinhydrolysate agar 100ml, add the HRBC liquid 5ml that the people prepares, slowly shake up and promptly get human red blood cell culture medium, the aseptic plate of the system of inclining, general plate by the 9cm diameter adds the 25ml nutrient solution and gets final product.
Embodiment 3, get and deposit the 3-7 days aseptic blood of carrying behind the slurry in the 10ml blood bank, isolate aseptic red corpuscle, with stroke-physiological saline solution washing 2-3 time, take the gimmick of rotating gently when washing under the room temperature at every turn, shake 20-30 time to same direction, and is firmly not excessive, in order to avoid haemolysis.Leave standstill the back and supernatant liquor is discarded, promptly get HRBC liquid with aseptic straw.
Get ox meat extract powder 24g, caseinhydrolysate 58g, starch 15g, agar 34g, add 2620ml distilled water, transfer PH=7.5,15 pounds of 30 minutes sterilising treatment, get caseinhydrolysate agar, get that sterilising treatment is chilled to 50 ℃ caseinhydrolysate agar 80ml, add the HRBC liquid 8ml that the people prepares, slowly shake up and promptly get human red blood cell culture medium, the aseptic plate of the system of inclining, general plate by the 9cm diameter adds the 25ml nutrient solution and gets final product.
Claims (5)
1, a kind of preparation method of human red blood cell culture medium is characterized in that 15 pounds of 30 minutes sterilising treatment postcooling of caseinhydrolysate agar to 46-55 ℃, and be 5-20 by volume with HRBC liquid: 1 mixes.
2, according to the preparation method of the described a kind of human red blood cell culture medium of claim 1, it is characterized in that caseinhydrolysate agar is is 4-24% ox meat extract powder by weight percent, 38-58% caseinhydrolysate, the starch of 1-15%, the agar of 14-34% mix the hydrolysis of back adding distilled water and form.
3, according to the preparation method of the described a kind of human red blood cell culture medium of claim 2, when it is characterized in that hydrolysis the amount of adding distil water be four kinds of component gross weights of casein agar 20-28 doubly.
4, according to the preparation method of the described a kind of human red blood cell culture medium of claim 2, transfer PH=7.4-7.6 when it is characterized in that hydrolysis.
5, according to the preparation method of the described a kind of human red blood cell culture medium of claim 1, it is characterized in that HRBC liquid is that surplus red corpuscle with the stroke-physiological saline solution washing, leaves standstill after extracting blood plasma by people's venous blood in sterile flask, abandoning supernatant forms.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN98110037A CN1079435C (en) | 1998-01-21 | 1998-01-21 | Method for preparing human red blood cell culture medium |
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|---|---|---|---|
| CN98110037A CN1079435C (en) | 1998-01-21 | 1998-01-21 | Method for preparing human red blood cell culture medium |
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| CN1224061A true CN1224061A (en) | 1999-07-28 |
| CN1079435C CN1079435C (en) | 2002-02-20 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103261404A (en) * | 2010-12-16 | 2013-08-21 | 默克专利有限公司 | Dry granulated cell culture media |
| CN104928240A (en) * | 2015-07-01 | 2015-09-23 | 浙江元太生物科技有限公司 | Preparation method of autologous or allogeneic red blood cell culture medium |
| CN108103033A (en) * | 2017-12-07 | 2018-06-01 | 武汉博威德生物技术有限公司 | A kind of fixed point remodeling method of rna virus cdna group |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5464755A (en) * | 1994-04-29 | 1995-11-07 | Biolog, Inc. | Microbiological medium and method of assay |
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1998
- 1998-01-21 CN CN98110037A patent/CN1079435C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103261404A (en) * | 2010-12-16 | 2013-08-21 | 默克专利有限公司 | Dry granulated cell culture media |
| CN105274047A (en) * | 2010-12-16 | 2016-01-27 | 默克专利有限公司 | Dry granulated cell culture media |
| CN103261404B (en) * | 2010-12-16 | 2017-06-09 | 默克专利有限公司 | Dry particulate cellular culture medium |
| CN105274047B (en) * | 2010-12-16 | 2019-03-01 | 默克专利有限公司 | Dry particulate cellular culture medium |
| CN104928240A (en) * | 2015-07-01 | 2015-09-23 | 浙江元太生物科技有限公司 | Preparation method of autologous or allogeneic red blood cell culture medium |
| CN108103033A (en) * | 2017-12-07 | 2018-06-01 | 武汉博威德生物技术有限公司 | A kind of fixed point remodeling method of rna virus cdna group |
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| CN1079435C (en) | 2002-02-20 |
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