UWSP COLLEGE OF NATURAL RESOURCES BUILDING
AUTOCLAVE OPERATIONS AND INSTRUCTIONS
INTRODUCTION
Any materials considered to be a biohazard or Potentially Infectious Material (PIM) must be disposed of in accordance with local, county and state regulations as detailed in OSHA Standards – 29 CFR, Standard Number 1910.1030 and WI NR Chapter 526. The PIM must either be rendered non-infectious through a means of sterilization or must be incinerated by a contractor licensed to handle infectious waste.
Steam sterilization is a dependable procedure for the destruction of all forms of microbial life. The autoclave uses saturated steam under a pressure of ~15psi to achieve a chamber temperature of at least 121 degrees Celsius (250 degrees F) for a minimum of 30 minutes.
Biohazards or PIM must be collected in a leak-proof, puncture-resistant container that is identified with red or orange “BIOHAZARD” label in the lab where they are generated.
Examples: Biohazard bag within a plastic bucket; corrugated cardboard “Burn Box” lined with a heavy duty plastic liner. The collected materials must have the leak proof bag sealed and must be transported to the Biology Dish Room, TNR 193D, on a cart or within secondary containment. The PIM will be sterilized by trained designated staff only.
Two methods of verification that sterility is achieved are used for all PIM:
1- A piece of autoclave tape should be affixed to each load of PIM processed through the autoclave. This tape is white, but upon reaching the sterilization temperature and holding that temperature for 15 minutes, the words “Autoclaved” and /or dark stripes appear upon the tape. This tape will usually indicate that your machine has functioned acceptably but the tape is not 100% reliable because the age of the tape and the conditions under which it is stored can sometimes effect a color change in the tape, yielding a false positive. 2- A microbial ampoule is a small poly container with vented cap that has an interior sealed glass vial containing the organism, Geobacillus stearothermophilus, and a purple media. One ampoule is placed into the autoclave with every load that is run. Upon completion of the sterilization cycle, the test ampoule should be removed, and incubated at 60 C. The length and method of incubation is determined by the manufacturer of the microbial ampoule so read the manufacturer’s literature carefully. Some brands of ampoules must be incubated in a water bath and read 48 hours later. Other ampoule brands require dry block incubation and may be read after 12 hours. If you use the wrong method of incubation, (putting a dry block ampoule into a water bath) you will have a false positive. The inner glass vial of any ampoule must be fractured so that the organism and indicator liquid are direct contact with the poly sides of the ampoule. This allows the incubator to most efficiently heat the ampoule. Failure to fracture the inner glass vial of the ampoule may lead to another false positive reading. If the bacillus inside the ampoule was NOT killed by the autoclave process, the organism grows, changes the pH of the liquid in the ampoule, and the vial’s liquid turns yellow and becomes cloudy. Therefore, after appropriate incubation of the vial, no color change of the purple media liquid would indicate that sterilization occurred because there was no microbial growth. The PIM load can be discarded after microbial verification of sterilization. If there is a color change of the ampoule’s media from purple to yellow, it means that sterility was not achieved and the load must be reprocessed through the entire cycle. 3- Once per week, a microbial control ampoule must be incubated without being sterilized to document that the ampoules used for verifications are viable. If the control ampoule does not show signs of growth, the test must be considered invalid and the PIM load must be re- sterilized.
AUTOCLAVE OPERATION – Consolidated Model SR-24A-PB
Personnel who operate the autoclave must have prior approval of TNR Building Environmental Coordinator. Operation approval is contingent on the following criteria:
- Documentation of Hepatitis B vaccination history. - Completion of UWSP General Safety Blood Borne Pathogen Safety training as provided by the Environmental Health and Safety office. - Completion and documentation of Autoclave Operation and Bio hazardous Spill Response Training as demonstrated by the Building Environmental Coordinator.
STEP 1 – Personal Protective Equipment
All operators must wear a buttoned lab coat and splash/impact resistant goggles.
STEP 2 – Load Preparation
All items to be sterilized must be placed in secondary containment, an approved autoclave tub. Tightly sealed bags should be cut or open slightly to assist with steam saturation. If the autoclave bag is tightly packed, add 250 ml of water to the inside of the bag. Four 17” wide by 20” long autoclave tubs will fit into the autoclave chamber for a full load. A small strip of autoclave indicator tape should be placed on each bag, basket or item.
A single microbial vial must be placed within the chamber for every load that is run. The microbial vial should be laying horizontally on the lowest shelf closest to the autoclave drain.
STEP 3 – Operators must put on heat resistant gloves before placing the autoclave tubs into the chamber. STEP 4 – Close autoclave door by engaging large lever, then turning door wheel to the right until it can no longer be turned. (Hand tight is sufficient.)
STEP 5 – Select cycle be depressing the button. The cycle options are as follows:
Color Cycle Description
Green Fast Exhaust Loads can be exhausted to atmospheric pressure quickly Yellow Fluid Liquids that will need to be reduced to atmospheric pressure very slowly to prevent boil-overs Blue Dry Loads of packs or instruments that require a drying time after all the steam are exhausted.
STEP 6 – Set sterilization time on MKII Control Panel by opening panel door and using thumbwheels to select the amount of time you want the load processed. You will have to estimate the weight of filled biohazard bags and add the total load weight, then us the e chart below as a guideline for the correct processing time.
CYCLE LOAD WEIGHT MINIMUM TIME COMMENTS
Fast 0 – 15 lbs 30 minutes Load = Small Fast 15 -25 lbs 50 minutes Load = Medium Fast 25 – 35 lbs 70 minutes Load = Large Fast 35 lbs or more 90 minutes Load = XLarge
Cycle time for loads of fluid filled containers is based on the largest container’s volume. If there are mixed loads, e.g.’ one 1000 ml flask, six filled square 18 mm test tube baskets (ea test tube holds about 20 ml) and two 500 ml flasks, you should select the processing time for the 1000 ml flask.
CYCLE EA CONTAINER VOLUME* MINIMUM TIME
Fluid 0 – 75 ml 25 minutes Fluid 75 – 250 ml 30 minutes Fluid 250 – 500 ml 40 minutes Fluid 500 – 1000 ml 45 minutes Fluid 1000 – 1500 ml 50 minutes Fluid 1500 – 2000 ml 55 minutes
Special note: fluid containers should not be tightly sealed and should be filled no more that ½ to ¾ full.
STEP 7 – Press red power button to start the autoclave cycle.
TO ABORT A CYCLE IN PROGRESS, PRESS THE RED BUTTON TO SHUT OFF POWER TO THE UNIT, then wait for the pressure gauge to reach 0 before attempting to open the door. Note: if a cycle is aborted prior to successful termination, it must be considered incomplete and the load must be reprocessed through the entire autoclave cycle.
STEP 8 – An end of cycle a buzzer will sound when the autoclave has successfully completed a cycle. To turn off the autoclave and buzzer, press the red power button.
STEP 9 – After the pressure gauge indicates 0 chamber pressure, operators must put on personal protective equipment: buttoned lab coat, splash/impact resistant goggles, and heat resistant gloves. The autoclave door can be opened by turning the door wheel to the left until it no longer can be turned. Stand away from the autoclave while raising the door lever to open the chamber and slowly swing the door ajar.
STEP 10 – Wait about two minutes with the door ajar before reaching in to unload the autoclave tubs to the designated spot.
STEP 11 – Remove the microbial verification ampoule from the autoclave. Wait ten minutes at ambient temperature. Then the inner glass vial of the ampoule should be broken using an appropriate tool. All ampoules should be incubated in the vertical position. Place the ampoule into the type of incubator that the ampoule manufacturer recommends.
STEP 12 – Remove PPE.
STEP 13 – Wash hands thoroughly with antimicrobial soap.
RECORD KEEPING
To document that all procedures have been followed, it is necessary to keep a log of the PIM loads that you run and the observations that you make. There is a clipboard in the autoclave room and can be handwritten IN INK.
Fill out the Autoclave Sterilization Log Sheet as per instructions. Enter data to fill in each blank writing neatly and staying within the form’s lines. If you make an error, do NOT erase, white out, or scribble over the error. Draw one line through the error, put your initials by the error, then the correct information on the next line.
Visually verify that sterilization met the critical standard by noting on the log sheet that the indicator tape changed from white to white with “autoclaved” printed in black. Enter this visual verification on the Autoclave Sterilization Log.
After incubation of the microbial ampoule, check the test ampoule you sterilized against the weekly control ampoule. Enter your observations on the AUTOCLAVE VERIFICATION-SPORE TEST form. These forms, like most safety and compliance records, must be kept for a period of thirty years.