AsepticTechnique
Background:
Intheearlydaysofsurgerythe mostimportant considerationwhenchoosingasurgeon was how fast could he performthesurgery. Therewasno anesthesia orsterile operating rooms. Surgery wasmessy and often patients died of shockorpost-operative infections. Where didthe infections come from you might ask?Ourcurrentunderstandingofthe ubiquitous nature of microorganismsandthe role that some play in disease wasunknown at the time, so surgeons had noconceptthatbynotproperlysterilizing their instruments orpreparingthepatientthey were contributing to the spread ofdiseaseanddeath.It wouldnotbe untilthe mid to late 1800’s before our moderngermtheory came to be widely acceptedandtheconsequencesofnotusing aseptic technique were fully understood.
The word aseptic literally means withoutsepsis.Sepsisisawordthat meansinfection. Therefore if a surgeon waspracticingaseptictechniquetheywould beoperatinginsucha manner asnottotransmit aninfectious agent orcauseaninfectiontooccur.Intoday’soperatingrooms, aseptic techniques are strictlyadheredsoastopreventinfection.Thetechniques include theuse of antiseptics,which are substances when applied to the tissues of the body prevent infections, proper sterilization of surgical instruments, proper handwashing, and the use of barriers such as gloves. These measures have no doubtreduced suffering and saved many lives.
Inthelaboratorythe aseptic technique islessconcernedwiththe transmission of
infectious agents asitiswiththecontamination oflaboratoryculturesandpersonnel working inthelab.
Contamination istheintroductionofanunwanted organismor organisms into aculture,environmental space, otherorganism(such as your lab partner) oryourself.Youwillbe working withavariety of bacterialculturesduringthe courseofthe semester.Itis importantthat you work with the organisms safelyand not contaminate them or yourself.
To accomplish thisyouwillneedtokeepa few things in mind.
You must be aware of the sources ofcontamination. The first is theenvironment. We are literally living in asea of microorganisms. There arebillions of bacteria on mostenvironmental surfaces as well as fungalspores, viruses and perhaps a fewprotozoan parasites.If these organismswere to enter your culture they maybegin to grow and your pure culture willno longer be pure. In the laboratoryexercises wewillbe conducting itisimportant thatwehavepureculturestoachieveproperresults.
A second source of contamination iswhatwerefertoascross contamination.This occurs when organisms fromoneculture areintroduced into another.Thiscan be deliberate or unintended, but theresultisthe same, a mixed culture. Youwillneedtosee that this doesnothappen.
Thelastthingto remember isthatifyouspillanyofyour organisms ontoyourlab bench or other surface you must
properlycleanupthespill immediately.While the bacteria we will be workingwithare not likely tocause problems ifproperly handled, they have the potential tocauseinfectionsifyou contaminateyourself with them.Remember safetyfirst.
Basic Principles of Aseptic Technique:
Thereareafewbasicconsiderationsyou needtokeepin mind whenworkingwithmicroorganisms. If you learn theseprinciplesandapplythem,youshould have no problemkeeping your cultures pureandachievinggoodresultsinthe lab. It should be noted that these principlescanbeextendedtothemedical andhealthcarearenas,aswellas the kitchen, bathroom, and to someextentthe bedroom.
Thefirstprincipleisthatyoushould consider EVERYTHINGto becontaminated with microorganisms.Thisisnot meant tofrightenyou,asmost bacteria and fungi you will encounterinyourdaytodaylifeareharmless toyou,butifthesecrittersarenot accounted for, they couldcontaminate a laboratoryculture.In themedical world, some of these organismsifgiventheopportunitycouldcausean infection.
Thesecondprincipleistoneverwork with microorganismunless you knowthatthetoolsyouare working with andthe mediumyou are using are sterile.Inthislabyoucan assume thatthe media Iwill supply is sterile,buttherearetimes it may becomecontaminated, solookitoverbeforeyouuseit.Culture mediumshould not be fuzzy or come withbacterialcolonieson the surface.
The third principle is thatculturesshouldbe axenic, whichmeans they containonly one species of bacteria. On occasion mixedculturesmay be used fora very specific reason. Since most of thecultures areaxenic when given to you,theyshould remain thatway.
Aseptic Technique
Now that you have a background concerning the basic principles of aseptic technique, itis time tooutlinehowyouaretoapplytheseprinciplesin the laboratory.
1)the first thing you do upon entering thelaboratoryistowipedownyour work area with disinfectant. Thiswillnot eliminate allthemicroorganisms on the lab bench,but remove most, particularly anypotential infectious agents.
2)thesecondthingyoudoistopaycareful attention toall instructionsgiven concerning the laboratory proceduresyouwillbeaskedtoperform.
3)whenyouactuallybegintowork with the microorganisms, you needto followingthe following steps andprotocols:
4)light your Bunsen burner. Be sure it is in a safe place and will not cause afire.Youwillbeinstructedontheproper use of the Bunsen burner.
5)obtain your inoculating loops and flamethem. The entire wire portionoftheloopmustbeallowedtoremain in theflame until it glowsred.Theloopshouldbeheldona slightangle.seeFigure1
Figure 1 Flaming the loop
6)once the loop is flamed, it is sterile.DO NOTtouch the loop, set it down on the lab bench, or allow it to comeintocontactwithanything.Itisnow readyto be placedintothe culture toremove a small amount of theorganisms.
7)carefullyremovethecapontheculture. DO NOT set the cap ontothelabbench;holdontoitinsuchamanner asitisheldrightsideup.
8)pass the mouth of the culture tubethrough the flame several times.Theideahereisto remove anymicroorganisms on the lip of the tubeand to warm the tube. DONOTleave the tube in the flame until itglows red. If you do, you will not likethe results.Also, DONOT holdthe tube vertical. Always keep the tube on a slight slant.
9)oncethetubehasbeenpassed throughthe flame, insertthesterileloop into the culture tube and downinto the culture.Gently remove a
loopful of the culture. The loop isnow referred to as being charged andcontains thousands ofmicroorganisms. DO NOTtouchanything with the loop or shake it around.
10)before putting the cap back onto theculture tube, pass the mouth of thetubethroughthe flame several times.Replace the cap.
11)at this point, you are ready to transferthebacteriaonthelooptoamicroscope slide or to culturemedium– broth or solid medium.
12)afterthetransfer,you must flame theloopagainto remove any remainingmicroorganisms. Once flamed theloopcanbesetaside.Remember itis hot.DO NOTtouch the loop ortouchanythingwithitthatcouldbe burned,i.e.yourlabpartnerorlabmanual, etc…
13)each time youaregoingintooroutofaculturetoeither removeorganisms or to inoculate themedium, you must flame the loopand the mouth of the tube.
14)after you have finished all the transfers and arereadytoleavethelaboratory,wipedownyourwork areawithdisinfectantandwashyour hands.Itisgoodpracticetowash yourhandsevery time youleavethelaboratory.Youreallydonotwant totakeanyofthe microorganismswithyou.Theydonot make goodpets.
NOTES:
This exercise is designed to help you practiceyouraseptictechnique.
1)obtain two tubes of mediumfromthe instructor
2)labelthetubes#1and#2with your wax pencil
3)opentube#1andinsertyourloop into the tube without flaming itorthetubemouth.Thereisno needtobecarefulhere.Youmay evenallowthetubetoremain uncapped for severalminutes if you like
4)usingthe aseptic techniquedescribed above repeat the
processinstep3.This time becareful to flame the loop, andtube. Do not allow the tube toremain uncapped for any longerthan necessary to place the loop into the mediumand remove it.
5)place both tubesintoarackasinstructed. The tubes will beincubated overnight andexamined for growth.
Day Two
1)examine the tubes for growth.Growthwillbe evidenced bycloudiness in the medium.
2)recordyourresultsinTable1.
Table 1 - Results
Tube #1 / Tube #2Wasthereadifference between thetwotubes?
Iftherewasadifference,howcanthisbeexplained?