CN111426532A - Bacterial wax block preparation method and method for positive quality control of bacterial wax block as acid-fast staining - Google Patents

Bacterial wax block preparation method and method for positive quality control of bacterial wax block as acid-fast staining Download PDF

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CN111426532A
CN111426532A CN202010271618.7A CN202010271618A CN111426532A CN 111426532 A CN111426532 A CN 111426532A CN 202010271618 A CN202010271618 A CN 202010271618A CN 111426532 A CN111426532 A CN 111426532A
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bacterial
acid
agar
bacteria
liquid
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余光银
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Peking University Shenzhen Hospital
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/36—Embedding or analogous mounting of samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/44—Sample treatment involving radiation, e.g. heat
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/84—Systems specially adapted for particular applications
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
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Abstract

The invention provides a method for preparing bacterial wax blocks, which is convenient for bacterial preservation and subsequent detection. The preparation method of the bacterial wax block comprises the following steps: inactivating the culture solution inoculated with the target bacteria; centrifuging the culture solution, and removing the centrifuged supernatant to obtain a bacterial sample liquid; mixing the bacteria specimen liquid and liquid agar at the temperature of 60-66 ℃ according to a set proportion, and waiting for the agar to solidify after mixing; and (3) cutting the solidified agar block into a set size, and embedding the agar block by using paraffin as an embedding agent to prepare the bacterial wax block. The invention also provides a method for positive quality control of the acid-fast staining by using the bacterial wax block, which aims to solve the defect of incomplete quality control of the acid-fast bacillus in the prior art.

Description

Bacterial wax block preparation method and method for positive quality control of bacterial wax block as acid-fast staining
Technical Field
The invention belongs to the technical field of pathology, and particularly relates to the field of histological wax lump simulation and a method for positive quality control of acid-fast staining.
Background
In clinical pathological diagnosis, to determine whether a lesion caused by tubercle bacillus infection is diagnosed, the 'gold standard' for diagnosis is found in body fluid and related tissue sections of a patient. Bacteria of the genus Mycobacterium such as Mycobacterium tuberculosis are acid-fast bacteria, and the tubercle bacillus is usually stained by the Ziehl-Neelsen method. The Acid Fast Bacillus (AFB) staining analysis of sputum smear has a long history for tuberculosis diagnosis, but the quality control of the acid fast bacillus of tissue sections is not perfect, and due to the requirement of quality control, a positive control needs to be set for special staining to eliminate false negative so as to prove that the staining result is reliable. In acid-fast staining, the positive rate of acid-fast bacilli is very low, and the positive rate is required to be very low. If the positive control is not provided and no positive bacteria are found, it is not advisable to determine the cause, for example, whether the tissue itself is truly negative or fails to stain, and it is also possible that the tissue containing tubercle bacillus has been excised. We can use pathological wax masses known to be positive as controls, but such control tissues are very difficult to find due to the very low positive rate of acid-fast staining. Moreover, after the tissues are subjected to pathological routine treatments such as fixing, dehydrating, embedding, slicing, dewaxing and the like, the bacteria content in the slices is greatly lower than that of fresh samples such as sputum and the like, so that a lot of time is spent on searching positive bacteria in the tissues. For tissues with low bacterial content, it is difficult to ensure that each tissue has bacteria by serial sectioning, and therefore, it is not suitable for use as a control tissue. However, the smear method is not preferred because it is not convenient to add test tissues and it is difficult to repeat the process.
The cell wax block is prepared by embedding loose cells or tissue fragments with paraffin, is convenient for storage and subsequent detection (such as special dye, IHC, molecules and the like), and the prior art is lack of a preparation method for the bacterial wax block.
Disclosure of Invention
One object of the present invention is to provide a method for preparing bacterial wax blocks, which facilitates the preservation and subsequent detection of bacteria.
It is another object of the present invention to provide a method for better positive quality control of acid fast staining.
In order to solve the first problem, the invention provides a method for preparing bacterial wax block, comprising the following steps:
step A, inactivating a culture solution inoculated with target bacteria;
step B, carrying out centrifugal treatment on the culture solution, and removing the centrifuged supernatant to obtain a bacterial specimen liquid;
step C, mixing the bacteria specimen liquid and liquid agar at the temperature of 60-66 ℃ according to a set proportion, and waiting for the agar to solidify after mixing;
and D, dividing the solidified agar block into set sizes, and embedding the agar block by using paraffin as an embedding agent to prepare the bacterial wax block.
The invention has the advantages that the method or the technology for preparing the bacterial wax block does not exist in the prior art, the inactivation step is adopted in the process, the risk of infection is avoided, the agar is solid at normal temperature, the liquid agar and the bacterial specimen liquid are selected and mixed to facilitate the dispersion of bacteria, the agar plays a supporting role after solidification, the subsequent observation is facilitated, and the strain can be conveniently stored or detected after the bacterial wax block is prepared.
In one embodiment of the present invention, the inactivation mode in step A is inactivation by adding 75% alcohol to the culture solution. The addition of 75% alcohol to the broth is intended to inactivate the bacteria, otherwise there is a risk of infection. Addition of 10% formalin also has a bactericidal effect, but formalin has an irritating taste and is not as convenient as alcohol. The high temperature inactivation is not convenient and causes the change of the bacterial form, so the method is not selected.
In an embodiment of the present invention, step B1 is further included between step B and step C: and heating the bacterial specimen liquid to 50-66 ℃. The liquid heating of the bacterial specimen is prepared for the subsequent mixing of liquid agar, and when the liquid of the bacterial specimen is mixed with the liquid agar, if the temperature of the liquid of the bacterial specimen is too low, the liquid agar is rapidly solidified, so that the liquid of the bacterial specimen and the liquid agar cannot be uniformly mixed.
Preferably, the liquid agar and the bacterial specimen liquid are heated at a temperature of 65 ℃. This temperature is for keeping agar's liquid state on the one hand, convenient and bacterium sample liquid mixing, be unlikely to the structure of the too high destruction bacterium of temperature simultaneously, on the other hand bacterium sample liquid adopts and agar-agar same temperature, the temperature difference has not been had, the two is cooled down in step when mixing bacterium sample liquid and liquid agar so, avoid when bacterium sample liquid temperature is less than liquid agar, each part of liquid agar because contact the low temperature bacterium sample liquid after solidify the difference fast, the problem that the echelon solidifies appears, make the bacterium can not evenly distributed in the agar-agar block after solidifying. The liquid heating mode of the liquid agar and the bacterial sample is preferably water bath heating, the temperature of the water bath heating is easy to control, and the temperature of the liquid agar and the bacterial sample liquid can be stabilized at the selected temperature.
In one embodiment of the invention, in order to ensure that the bacterial mass of the prepared bacterial wax block after slicing is suitable and sufficient for observation, the bacterial sample and the liquid agar are mixed according to equal volume proportion.
The technical scheme adopted by the invention for solving the second problem is as follows:
a method for positive quality control of a bacterial wax pellet as an acid fast stain, comprising the steps of collecting acid fast bacteria, transferring the acid fast bacteria to a carrier observable under a microscope, and staining against the acid fast bacteria, wherein the carrier is a bacterial wax pellet made in the first problem.
In one embodiment of the invention, the collecting of acid-fast bacteria in the step comprises a sub-step of pleural effusion culture; the pleural effusion culture is to place the pleural effusion inoculated with acid-fast bacteria in a carbon dioxide incubator at the temperature of 24-26 ℃ after being sealed for culture for 40-52 h.
Preferably, the temperature of the incubator is 25 ℃.
Preferably, the standing time of the pleural effusion in the incubator is 48 h.
The invention takes the pleural fluid as the culture medium, spreads by utilizing the natural invasion and growth of bacteria, has good effect, effectively disperses large bacterial colonies, almost tiles the bacteria, rarely has the condition of more than 2 layers, greatly increases the bacterial amount, is very beneficial to observation, is suitable for mass production, and avoids the defect that the sputum smear method can not be repeatedly produced. Such bacterial wax clumps are ideal positive quality control materials.
Drawings
FIG. 1 is a microscopic image of a tuberculosis bacterial wax block slice prepared by a pleural effusion culture method and magnified by 600 times.
FIG. 2 is a microscopic image of a tubercle bacteria wax block slice prepared by a centrifugal culture method and magnified by 100 times.
FIG. 3 is a microscopic image of a tubercle bacteria wax block slice prepared by a blending method with magnification of 100 times.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.
1.1 tubercle bacillus source tubercle bacillus cultured in the clinical laboratory of the institute of My (Shenzhen hospital of Beijing university) for about 60 days and confirmed by acid-fast cold-staining method was used.
1.2 carbon dioxide incubator, Ebende centrifuge, sakura dehydrator, Meikang embedding machine, Saimeifei H340E paraffin slicer, agar, acid-fast staining reagent (phenol alkaline pinkish solution: ① 5g of basic fuchsin is dissolved in 100ml of 95% ethanol to make basic Pinhong ethanol solution; ② phenol is put into a incubator at about 50 ℃ to make it dissolved, 5ml of phenol is weighed and mixed with 95ml of distilled water completely, ③ 10ml of basic fuchsin ethanol solution is mixed with 90ml of 5% phenol aqueous solution to make phenol alkaline pinkish solution).
1.3 preparation method of bacterial wax block
1.3.1 Collection of bacteria. (in clinical laboratory the bacterium cultured by inoculation was confirmed to be an acid-fast bacterium tuberculosis by "Cold staining method")
A pleural effusion culture method is utilized, wherein a 50ml centrifuge tube is taken, 10ml pleural effusion (within 1 h) is poured in advance, the operation is carried out in a biological safety cabinet, the cultured tubercle bacillus mass is picked into the pleural effusion by an inoculating loop, the inoculating loop is tightly covered, the inoculating loop is placed in an incubator at 25 ℃, the inoculating loop is taken out after 48h, 40ml of 75 percent alcohol is poured, the inoculating loop is soaked and sterilized for 30min, the inoculating loop is centrifuged at 2000rpm for × 10min, and the supernatant is poured out for standby.
1.3.2 taking about 1-2 g of the prepared agar by using a key, putting into a 50ml centrifuge tube, putting into boiling water, boiling for 30min with the lowest fire, dissolving, and putting into a 65 ℃ water bath for later use.
1.3.3 the specimen centrifuge tubes prepared by the centrifugation method, the mixing method and the hydrothorax culture method are respectively placed in a 65 ℃ water bath and preheated for about 1 minute.
1.3.4 add liquid agar equal to the liquid, mix gently quickly, and set in ice water.
1.3.5 after the agar is completely solidified, one end of a bamboo tip is inserted along the side of a test tube, the agar block is carefully withdrawn, cut into a proper size of about 3 × 5mm, placed in an embedding box, and then conventionally processed by a machine, dehydrated and embedded to prepare wax blocks, the yield of the single tube bacterial wax blocks is obviously different, 7 blocks are obtained by a centrifugal method, 6 blocks are obtained by a uniform mixing method, and 38 blocks are obtained by a pleural effusion culture method.
1.3.6 to further evaluate the number and the dispersion degree of bacteria, 6 bacterial wax blocks are randomly taken out by each method, after the blocks are trimmed, the wax blocks are cut into slices at intervals of 100 mu m, 5 wax blocks are cut into 5 wax blocks, 30 wax blocks are cut into a total slice by each method, and after the wax blocks are stained, the wax blocks are randomly and averagely submitted to 5 doctors for evaluation, and 6 wax blocks are placed on each doctor. Staining was done according to Ziehl-Neelsen staining modification (modified Wade-Fite). The method comprises the following specific steps:
1.3.6.1 slicing into 4 μm, baking at 70 deg.C for × 30 min.
1.3.6.2 slices are dewaxed for 2 times in the gasoline and turpentine mixed liquid, and each time lasts for about 5-10 min.
1.3.6.3 the remaining liquid around the cut pieces was wiped dry with absorbent paper, but the cut pieces should remain slightly moist.
1.3.6.4 rinsing with running water for 1 min.
1.3.6.5 and adding phenol alkaline red solution dropwise to dye at room temperature for 20-30 min.
1.3.6.6 running water washes off excess dye liquor.
1.3.6.720% sulfuric acid.
1.3.6.8 flushing with running water for 5 min.
1.3.6.9 the Mayer hematoxylin lightly stains the nucleus.
1.3.6.10 rinsing with running water for 10 min.
Oven drying at 60-70 deg.C at 1.3.6.11, transparent with xylene, and sealing with neutral gum.
Referring to the attached figure 1, a microscopic image of a tubercle bacteria wax lump slice prepared by a pleural effusion culture method and magnified by 600 times is shown. The acid-fast bacteria (tubercle bacillus) is red in the figure, and the nucleus is blue. The prepared tubercle bacillus cell wax block can see about 3 small tubercle bacillus clusters and dispersed tubercle bacillus in each high-power visual field, and completely meets the requirement of positive quality control tissues. Due to the cell components (or the light red background after cell lysis), the volume of the wax block prepared after centrifugation is obviously increased, which is about 6 times of the previous volume, and the method is very suitable for preparing a large amount of control tissues. And due to the cell components, the background is provided for the acid-fast bacteria, and the macroscopic form and the microscopic form can be observed more conveniently.
The positive control tissue requires that each section is positive and convenient to observe, so we need to know the total amount and distribution of bacteria in the prepared bacterial wax block, the bacterial colony in the culture method is small and thin in the high power field, for counting the bacterial colony number, we stipulate that the number of the accumulated bacteria is more than or equal to 30 to be one bacterial colony, each section is 20 high power fields (40 ×), A, B, C, D, E5 doctors count 6 sections per person, and calculate the average bacterial colony number as shown in the following table, and the average bacterial colony number is about 3 bacterial colonies in each field.
Table 1.
Average pellet count Ab C D E Total average (%)
Hydrothorax culture method 68.463.072.469.871.469.00
The pellet is too thick to facilitate observation of the bacteria. We counted the layering of bacteria to indicate the thickness of the pellet, and basically the layering of the pellet is 1-2 layers, which is very beneficial to the observation. Referring to the attached figures 2 and 3, the bacterial wax blocks prepared by the centrifugal method and the mixing method after cultivation are large tubercle mycoses under slice observation, bacteria are overlapped and difficult to observe, and each visual field can not be ensured to be seen at high power.
In practice, to ensure successful staining, it is necessary to add positive control tissue. However, in the pathology department, it is a difficult problem to find a control tissue which is simple, convenient, stable and reliable to manufacture, and the cultured tubercle bacillus is used for manufacturing the bacterial wax block by adopting a pleural effusion culture method, so that the bacterial wax block can be used as a reliable and practical acid-resistant positive control, and the method can help some pathology departments which are not easy to obtain the acid-resistant positive control tissue.

Claims (10)

1. A method for preparing bacterial wax blocks is characterized by comprising the following steps:
step A, inactivating a culture solution inoculated with target bacteria;
step B, carrying out centrifugal treatment on the culture solution, and removing the centrifuged supernatant to obtain a bacterial specimen liquid;
step C, mixing the bacteria specimen liquid and liquid agar at the temperature of 60-66 ℃ according to a set proportion, and waiting for the agar to solidify after mixing;
and D, dividing the solidified agar block into set sizes, and embedding the agar block by using paraffin as an embedding agent to prepare the bacterial wax block.
2. The method for preparing bacterial wax blocks according to claim 1, wherein the inactivation mode in step A is to add 75% alcohol into the culture solution for inactivation.
3. The method for making bacterial wax blocks according to claim 1, wherein the step between the step B and the step C further comprises the step B1: and heating the bacterial specimen liquid to 50-66 ℃.
4. The method for preparing bacterial wax block according to claim 1, wherein the bacterial specimen liquid and the liquid agar are mixed in equal volume ratio in step C.
5. The method of claim 3, wherein the liquid agar and the bacterial specimen liquid are heated to 65 ℃.
6. A method for positive quality control of bacterial wax pieces as acid fast stains comprising the steps of collection of acid fast bacteria, transfer of acid fast bacteria to a microscopically observable carrier and stain against acid fast bacteria, wherein the carrier is a bacterial wax piece made in claim 1.
7. A method according to claim 6, wherein the acid fast bacteria collection step comprises the sub-step of hydrothorax culture;
the pleural effusion culture is to place the pleural effusion inoculated with acid-fast bacteria in a carbon dioxide incubator at the temperature of 24-26 ℃ after being sealed for culture for 40-52 h.
8. A method according to claim 7, wherein the temperature of the incubator is 25 ℃.
9. A positive quality control method of bacterial wax as acid fast stain of claim 7, wherein the resting time of the pleural fluid in the incubator is 48 h.
10. A method according to claim 6, wherein the acid-fast bacteria is acid-fast bacilli tuberculosis.
CN202010271618.7A 2020-04-09 2020-04-09 Bacterial wax block preparation method and method for positive quality control of bacterial wax block as acid-fast staining Pending CN111426532A (en)

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Cited By (1)

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CN113358447A (en) * 2021-04-21 2021-09-07 广州金域医学检验中心有限公司 Fungus cell positive wax block and preparation method and application thereof

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