1. Necessity:
①Sterilization effect monitoring method is highly specialized, do not know the results of the test;
②new wards, new disinfection equipment, new disinfectants should be done to monitor the effect.
3 special objects, special needs, must do effect monitoring. Such as pre-transplantation, ICU wards, critically ill patients.
2. Scientific: scientific experimental design, skilled technology, reliable results, accurate analysis, results evaluation.
3. Normative: based on authoritative norms, in line with its basic principles. Such as the determination of the timing of the test, the selection of standard strains, the number of specimens, the number of repetitions, experimental methods and other established.
Second, the classification of monitoring methods:
1, according to the nature of the detection method:
① Physical methods: measurement of pressure sterilization temperature, measurement of ultraviolet intensity, etc.;
② Chemical methods: a variety of chemical indicator card;
③ Biological methods: thermophilic / chytridiomycetes sterilization effect of the monitoring of the number of natural bacterial viability monitoring;
2, according to the nature of disinfection and sterilization objects:
① air disinfection:
② surface disinfection:
3. According to the specific disinfection objects:
① Pressure Steam Sterilization:
② a variety of instruments:
③ chemical disinfectants:
④ ultraviolet lamp sterilization effect:
⑤ hand and skin Disinfection effect:
⑥Air disinfection effect:
⑦Object surface disinfection effect:
Three, the necessary conditions:
1, professional laboratories:
2, the necessary equipment and devices:
3, the choice of experimental methods:
4, skilled experimental techniques:
four, the Ministry of Health on the 500 The Ministry of Health of the quality of infection management in hospitals with more than 500 beds stipulates:
(1) hospital infection rate ≤ 10%; sterilization incision infection ≤ 0.5%
(2) underreporting of hospital infections ≤ 20%;
Surveys of the sample size of not less than 10% of the number of annual monitoring of the number of patients
(3) hospitals must be disinfected and sterilized effect of regular monitoring.
Sterilization pass rate must reach 100%
(4) disinfectants and sterilants in use should be biological and chemical monitoring. Disinfectant quarterly biological monitoring, bacterial content must be <100cfu/ml, shall not be detected pathogenic microorganisms; sterilizing agent monthly test, shall not be detected microorganisms. Chemical monitoring should be based on the performance of disinfection and sterilization agent regular monitoring.
(5) pressure steam sterilization for process, chemical and biological monitoring. Ethylene oxide must be process monitoring per pot, chemical monitoring per package, and monthly biological monitoring. Pressure steam pots must have a B-D test on the first pot of each day.
(6) ultraviolet lamp intensity monitoring: quarterly; new lamp 30W ≥ 90?W / cm2, old lamp ≥ 70?W / cm2
(7) gastroenteroscopy and other diagnostic and treatment equipment disinfection standards shall not detect pathogenic microorganisms; laparoscopes, arthroscopes, etc. should be sterilized and processed, and no microorganisms shall be detected.
(8) into the human body tissue, blood, organs of medical supplies should be sterilized, shall not detect pathogenic microorganisms; contact with mucous membranes of medical supplies bacterial total ≤ 20cfu / g (or 100 cm2); contact with the skin of the total bacterial count of medical supplies ≤ 200cfu / g (or 100 cm2), shall not detect pathogenic microorganisms.
(9) Salmonella shall not be detected on the surfaces of objects and hands of health care workers in the room with mother and baby, baby room.
(10) hospitals of various types of environmental air, object surfaces, health care workers hand bacterial count standards:
Environmental categories of air, object surfaces, health care workers hand
cfu/cm3 cfu/cm2 cfu/cm2
I Class I laminar flow clean operating room, ≤10 ≤5 ≤5
clean wards
II class General operating rooms, maternity wards, ≤200 ≤5 ≤5
baby rooms, general isolation wards
Ⅲ pediatrics, gynecological examination rooms, ≤500 ≤10 ≤10
injections, drug exchange, treatment,
emergency room, laboratory, general wards
Ⅳ infectious diseases and wards - - ≤15 ≤15
V. In order to ensure that the hospital disinfection and sterilization is reliable, the hospital disinfection and sterilization effect should be carefully monitored
1, there are many factors affecting disinfection and sterilization, making it difficult to ensure the effect of disinfection and sterilization.
2, sterilization effect of biological monitoring of professional, difficult to create the conditions, the results of the evaluation of the difficulty of the long experimental cycle.
3, chemical test reagent method is more complex, the results are accurate; test paper method is simple and easy to implement, but the accuracy is poor.
Six, pressure steam sterilization effect of monitoring:
(a) BD test paper:
Check the state of the sterilizer process, and can not respond to the sterilizer use of the actual sterilization effect of the sterilized items. Found positive should be carried out sterilizer performance check debugging.
(ii) indicator tape:
Indicates whether the sterilization process, and can not reflect the actual sterilization effect of sterilized items.
(c) chemical indicator card:
To detect the sterilization effect of each package of sterilized articles. It is recommended that each packet of sterilized articles in the center of a piece of chemical indicator card, to be sterilized, depending on the degree of discoloration (sterilizing effect) to decide whether to use.
(D) biological indicators for systematic monitoring:
The use of biological sterilization indicators to check the effect of pressure steam sterilization is the most scientific and reliable monitoring method. By the Chinese Academy of Preventive Medical Sciences Institute of Epidemiological Microbiology, Beijing Xin Sihuan Disinfection Technology Development Center joint production and marketing of thermophilic lipophilic Bacillus spores (SS1, K31) is an internationally recognized standard strain. It is now made into standard bacterial slices for checking the effect of pressure steam sterilization test.
w Characteristics of the strain:
1, the bacterium is aerobic bacillus, the bacterial propagation body is negative for Gram's staining is purple, the bacterial bacterial cells peacock green coloring. The bacterial propagation body has low requirements for culture medium, and grows well on bromocresol violet glucose peptone agar with a rough beige surface.
2, the optimal growth and reproduction temperature of 56-65 ℃, culture 24h can form colonies, 37 ℃ 24h can not see colonies.
3, this bio-indicator carrier for high-grade filter paper sheet, the amount of bacteria stained 5 × 105-106cfu / piece, encapsulated in a small paper bag, heat death time of 121 ℃, 3.9min positive, 19min negative; D10 value of 1.3-1.9min, in line with the United States Pharmacopoeia, the eleventh edition of the regulations Standard. The bacterium is non-toxic, thermoresistant and stable, stored in the refrigerator at 4 ℃ for one year without a significant decline in resistance, at room temperature (about 20 ℃) can be stored for 1 month.
w How to use:
1, the bacillus tablets will be used when the small paper bag containing slices of bacteria in the center of the sterilized items (the number of slices of bacteria placed in each pot in accordance with the provisions of the Ministry of Health).
2, after sterilization, and then aseptically removed from the paper bag of slices, put into the bromocresol violet culture tube. At the same time, the unsterilized bacterial pieces into another tube of medium for control.
3, 56 ℃ -60 ℃ culture, 48h observation results, the control tube is beige; if the color of sterilized bacterial slices of culture medium remains unchanged mauve, for negative (-), said sterilization is complete; such as yellow for positive (+), said sterilization is not complete.
w Media composition and preparation:
1, composition: tryptone 10g, glucose 5g, distilled water 1000ml, 1.6% bromocresol violet alcoholic solution indicator 1ml.
2, preparation:
(1) 1.6g bromocresol violet dissolved in 98.4ml of 96% alcohol solution shaking well;
(2) Dissolve the first three ingredients, mediate pH7.0-7.2, add the indicator and shake well;
(3) Dispense 5ml of each tube, sterilize it at 121℃ for 20min, and then put it into 4℃ refrigerator for storage and spare.
w Precautions:
1, to prevent the indicator in the alcohol volatilization and make the bromocresol violet content is too high (bromocresol violet content is too high after the inhibition of the growth of bacteria);
2, bacterial slices should be taken out of the culture in a timely manner after sterilization;
3, prohibit the slices of bacteria in contact with any disinfectant and radioactive sources so as not to affect the resistance.
Seven, chemical disinfectants disinfection effect monitoring:
(a) disinfectant effective content of the test:
1, mainly effective content of unstable disinfectants, such as peroxyacetic acid, chlorine disinfectants, etc. should be content test.
2, chemical reagent titration method is more complicated but the results are accurate; test paper method is convenient and fast, but the accuracy is poor. Dedicated chlorine test paper - more accurate; composite test paper (can be measured with peracetic acid, chlorine disinfectants) - less accurate. Glutaraldehyde test paper; test paper containing iodine, ozone, etc. is yet to be studied.
(B) chemical disinfectants using the number of contaminated bacteria in the solution detection
1, select and use a good neutralizer:
2, do: correspondence, concentration, proportion appropriate
3, neutralizer selection principles:
① itself has no effect on the bacterial
② really remove the bactericidal factor
3, the generation of the bacterial has no effect
3, the bacterium has no effect
4, the bacterium has no effect
4, the bacterium is the only one of the two disinfectants in the solution, the bacterial is the only one of the two disinfectants in the solution.
④Complete control
★Test grouping:
①Bacterial solution + drug solution culture
②(drug + bacterial solution)+neutralizing agent culture
③Neutralizing agent + bacterial solution culture
④(drug+neutralizing agent)+bacterial solution culture
⑤PBS+bacterial solution culture
⑥PBS culture
6 PBS culture
7 Neutralizer culture
8 Medium culture
1) Methods:
First step: Use a sterilized pipette with a capacity of 1 ml to draw up 1 ml of sterilized solution.
Step 2: Add the aspirated 1 ml of sample solution to 9 ml of diluent, the composition of this diluent preparation depends on the composition of the disinfectant solution, i.e., add a suitable neutralizer to the saline solution.
Disinfectant vat for soaking instruments Dilution tube Nutrient agar flatware
Figure 1 Kelsey and Maurer's proposed test of disinfectant solution in use
Step 3:
Take the above disinfectant dilution tubes to the laboratory, and don't allow more than 1h from sampling to test, followed by a 50-drops/ml volume dropper (out of the Available serum pipette with 0.02ml scale), suck the mixture, on the surface of each nutrient agar plate (the surface of the medium plate has no moisture), 10 drops (each drop amount of 0.02ml), the same drop two plates.
Step 4:
The two plates were incubated in two incubators with different temperatures; one was placed in a 37℃ incubator for 1 to 2 days; the other plate was incubated at room temperature around 20℃ for 3 to 7 days, after which the number of colonies on the two plates was counted, and divided by 2, which resulted in the average number of colonies on each plate.
2) Test results:
The presence of bacterial colonies on the plates after incubation indicates the presence of bacteria in the sterilizing solution. If a plate on the growth of 1 to 2 colonies, can be ignored, because the disinfectant solution is, after all, a chemical disinfectant, rather than sterilizing agent, so the culture of a very small number of live bacteria is normal. If a plate growth of 5 or more than 5 colonies, it should be noted that the disinfectant solution has been contaminated. From the number of colonies detected on the plate, the number of bacteria present in each milliliter of disinfectant solution can be calculated as follows:
◆ Since the disinfectant sample solution is diluted at 1:10, use 10 drops of a 50 drop/ml dropper. If 10 drops of the disinfectant/dilution mixture grow 5 colonies, it can be calculated that 250 viable bacteria are present in 1 ml of the disinfectant solution. In other words, with 10 drops on a plate, it is 1/5 of 1 ml, so the calculation is as follows:
5 (total number of colonies growing on a plate) x 10 (diluted 10-fold with disinfectant solution) x 5 (10 drops on a plate, which is only 1/5 of the disinfectant/dilution mixture) = 250 bacteria.
5 x 10 x 1 = 100 bacteria, i.e., the number of bacteria present per milliliter of disinfectant. If 20 colonies grow on a plate, there are 5 x 10 x 5 = 250 bacteria per milliliter of disinfectant solution, and such a large number of bacteria indicates that this disinfectant solution has failed and can no longer be used
(See Figure 2)
Figure 2 Contamination of bacteria on a plate
Few colonies of bacteria on each drop indicate that it can't be used any further
The presence of many Colonies on each drop indicate serious contamination
3) Reasons for the growth of bacteria in disinfectant solution:
The following reasons are common for the growth of bacteria in disinfectant solution:
1) The container has not been washed and disinfected without heating.
② Preparation of disinfectant solution, disinfectant and water is not accurate, so that the concentration of disinfection is too low to kill bacteria or inhibit bacteria.
3 ③ disinfectant solution stored for too long, has failed.
4) The disinfectant fails due to excessive organic substances in the disinfectant solution, which consume the disinfectant.
⑤ There are various disinfectant inhibiting substances in the disinfectant solution.
Eight, the detection of medical device sterilization effect:
Sampling time; after sterilization treatment, storage of samples within the validity period.
(A) routine monitoring
1, detection method: suture needles, needles, surgical blades and other pieces of medical equipment, each of 5 pieces, respectively, into 5 ml of sterile eluent. Syringes, on the other hand, take 5 pairs in 5 ml of sterile broth were pumped 5 times. Surgical forceps, forceps and other large medical equipment take 2 pieces, with a cotton swab repeatedly coated with sampling, the cotton swab into 5ml of sterile elution solution. Vibrate 80 times, suck 1ml inoculation dish for viable bacteria count, 37 ℃ culture 48h, calculate the number of colonies.
2, the results of the determination: no bacterial growth on the plate for sterilization.
3, Note: If the disinfection factor is a chemical disinfectant,
sampling solution should be added to the corresponding neutralizer.
(B) aseptic test
1, aseptic test preparation
(1) eluent and culture medium aseptic test: aseptic test 3 days before the inoculation of 1 ml of eluent in each of the required - anaerobic medium, respectively, to 30 ~ 35 ℃ and 20 ~ 25 ℃ incubation for 72h, there should be no bacterial growth.
(2) Positive control tube bacterial fluid preparation: the day before the test, take the fresh culture of Staphylococcus aureus (CMCC 26003), inoculated with 1 ring to the required - anaerobic medium, cultured at 30~35 ℃ for 16~18h, and then diluted with 0.9% sterile sodium chloride solution of 10cfu/ml standby. Take 1 ring of fresh culture of aerobic and anaerobic medium of Clostridium perfringens (CMCC 6494) and inoculate it into the same medium, incubate it at 30~35℃ for 16~18h, dilute it with 0.9% sterile sodium chloride solution for 10cfu/ml. Take Candida albicans (CMCC 98001) fungal agar medium slant fresh culture 1 ring, inoculated in the same medium, 20 ~ 25 ℃ culture 24h, with 0.9% sterile sodium chloride solution diluted 10cfu/ml ~ 10cfu/ml standby.
2, aseptic operation:
(1) take the suture needle, needle, blade and other small pieces of medical equipment 5 pieces of direct intrusion into the 6 tubes need - anaerobic culture tube (of which 1 tube as a positive control) and 4 tubes of mold culture tube. The amount of culture medium is 15ml/tube.
(2) Take 5 syringes and repeatedly draw 5 times in 5 ml of eluent, inoculate the anaerobic culture tubes (6 tubes, of which 1 tube is used as a positive control) and mold culture tubes (****4 tubes). Inoculation volume: 0.5 ml for 1 ml syringe, 1 ml for 2 ml syringe, 2 ml for 5-10 ml syringe, 5 ml for 20-50 ml syringe, 15 ml/tube for less than 2 ml of medium, 40 ml/tube for 5 ml of inoculum.
(3) surgical forceps, forceps and other large medical equipment to take 2 pieces of cotton swabs coated with sampling, into 5ml of sterile eluent, inoculated in the need - anaerobic culture tube (6 tubes, of which 1 tube for the positive control) and mold culture tubes (****4 tubes). The inoculation volume was 1ml/tube, and the amount of culture medium was 15ml/tube.
3, culture: anaerobic culture tubes and positive and negative control tubes were incubated at 30-35 ℃ for 5 days. Mold culture tubes and negative control tubes were incubated at 20～25℃ for 7 days.
4, the results of the judgment: positive control should have bacterial growth in 24h, negative control no bacterial growth, anaerobic culture tubes and mold culture tubes have no bacterial growth, can be judged as sterilization qualified.
★ Such as the need for - anaerobic culture tube and mold culture tube in any 1 tube show turbidity and prove that there is bacterial growth, should be re-sampled, respectively, retested 2 times, such as the tubes without bacterial growth can still be judged to be sterilized qualified.
5, notes:
(1) in the cleanliness of 100 class area, strict aseptic operation.
(2) The time of delivery after sampling shall not exceed 6h, and if the sample is kept at 4℃, it shall not exceed 24h.
(3) If the disinfection factor is a disinfectant, a neutralizing agent shall be added to the sampling solution.
(4) Sampling area, 100cm2.
(5) Set up positive and negative controls.
Nine, hand and skin disinfection effect test:
1, sampling time, sampling immediately after disinfection.
2, sampling method
(1) hand, hand surface finger heel to finger end (a hand of about 30cm2), sampling solution 10ml.
(2) skin, 5cm × 5cm . Sampling solution 10ml.
3, test method
Take 1ml of sampling solution for viable bacteria count, 37 ℃ culture 48h.
4, sampling results calculation:
The number of colonies on the plate X dilution multiples
Total number of bacteria (cfu/cm2) = ------------------------- --------
Sampling area (cm2)
5, the results of the judgment:
(1) I, II type of area staff, the total number of bacteria ≤ 5cfu/cm2, did not detect pathogenic bacteria for disinfection qualified.
(2) Type III area staff, the total number of bacteria ≤ 10cfu/cm2, did not detect pathogenic bacteria for disinfection.
(3) Category IV area staff, the total number of bacteria ≤ 15cfu/cm2, did not detect pathogenic bacteria as disinfection.
●Staff in the mother and baby room, baby room, neonatal room and pediatric ward shall not be detected with salmonella and other pathogenic bacteria.
6. Precautions:
(1) Sampling equipment should be sterile.
(2) The sampling solution should contain the appropriate neutralizer.
(3) The problem of setting the positive control.
(4) Sterilization time requirements, 3 minutes for surgical hand disinfection, 1 minute for hygienic hand disinfection, and 5 minutes for skin disinfection.
Ten, object surface disinfection effect monitoring
1, sampling time: sampling after disinfection treatment.
2, sampling method
With 5cm × 5cm sterilized specification plate, sampling area ≥ 100 cm2. door handles and other irregular surfaces are sampled directly with a cotton swab.
3, sampling solution 10ml (including neutralizer).
4, test method: the same
5, the results of the decision:
(1) I, II type of area, the total number of bacteria ≤ 5cfu/cm2, and did not detect pathogenic bacteria for disinfection.
(2) Class III area, the total number of bacteria ≤ 10cfu/cm2, did not detect pathogenic bacteria for disinfection.
(3) Category IV areas, the total number of bacteria ≤ 15cfu/cm2, not detected pathogenic bacteria as disinfection. Salmonella shall not be detected on the surface of objects in the mother and baby room, baby room, newborn room and pediatric ward.
Eleven, air disinfection effect monitoring
1, sampling time: after disinfection, sampling before operation.
2, sampling method:
(1) point:
Indoor area ≤ 30 m2, set up the inner, middle and outer diagonal 3 points, inside and outside the point of 1m from the wall; indoor area of >30 m2, set up the four corners and the center of the 5 points, the four corners of the point of 1m from the wall.
(2) Plate exposure method
Plates 9cm in diameter, sampling height 1.5m, exposure for 5min.
3, test method
The plate was incubated at 37℃ for 48h. The number of colonies was counted and the pathogenic bacteria were separated.
4. Calculation of the results of plate exposure method
50000N
Total number of bacteria (cfu/m3) = --------------------
A×T
A is the area of the plate (cm2); T is the time of exposure (min); N is the average number of colonies (cfu)
5, the results of the determination
(1) I, II type area, the total number of bacteria ≤ 10cfu/cm3, did not detect pathogenic bacteria for disinfection qualified.
(2) Class III area, the total number of bacteria ≤ 200cfu/cm3, did not detect pathogenic bacteria for disinfection.
(3) Category IV areas, the total number of bacteria ≤ 500cfu/cm2, did not detect pathogenic bacteria for disinfection.
6, Note: Before sampling, close the door and window, in the case of no one walks, static 10min for sampling.
Twelve, ultraviolet lamp intensity test:
1, ultraviolet light is invisible, ultraviolet light ≠ sterilization.
2, UV direct, refraction.
3, UV penetration is weak.
4, germicidal ultraviolet for the C-band, the center wavelength of 2537AO
5, the amount of ultraviolet germicidal agent: intensity X time
6, detection of ultraviolet lamp intensity method: ultraviolet intensity meter; ultraviolet intensity indicator card
7, the quality of ultraviolet lamps: glass without air bubbles, air lines; fast start; no flicker; long life; irradiation Intensity standard: 30W lamp new lamp ≥ 90?W / cm2; old lamp ≥ 70?W / cm2
8, UV intensity test from the light vertical 100cm irradiation directly read the intensity value.
Note: ① good protection: wear glasses, gloves, if necessary, wear a protective mask.
②Ultraviolet lamp is turned on after 5 minutes of stabilization, read the test values.
3 UV intensity changes with the quality of the lamp voltage, reflector finish and so on.
Thirteen, a few points of information and views:
1, the evaluation of disinfection technologies and methods of the problem:
1) chlorine-containing disinfectants replacement;
2) glutaraldehyde and o-formaldehyde;
3) dynamic disinfector effect evaluation;
4) ozone disinfection of the advantages and disadvantages of (oxidation is prominent);
5) Combination of air and surface disinfection;
◆Disinfection is active treatment and essential for anyone!
◆Healthy life is the most valuable!
◆In order to protect health and life, let's **** together for the development of disinfection!