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This Factsheetwill describethe following techniquesinvolved in culturing bacteria in the laboratory: . Safety in the microbiology laboratory; . Plating out; . Culture containers and instrumentsl . Inoculation and culture techniques; . Types of media and their preparation; . Anaerobic cultures.
PracticalTechniques Microbiology I: Culturing bacteria in
Factsheet No 71 'The control of bacteria' (April 2000) is also relevant to this topic. Introduction Basic safety rules for microbiology are listed in the appendix at the end
Bacteriological investigationsusually involve growing pure cultures in a nuffient medium under conholled conditions of temperature(usually 37"C) and pH (usually around pH7.\. The nutrient mediamust contain a sourceof: . carbon (e.g. glucose, another simple sugar, or a salt of an organic acid,e.g. sodium ethanoate); . nitrogen (usually amino acids, peptides,or ammonium salts); . mineral salts (commonly requiredpositive ions are those of calcium, potassium,sodium, and iron; commonly required negative ions are chloride, phosphate, and sulphate) . water. Also the bacteriamust have a sourceof energy: . Heterotrophs gain this by the oxidation of sugars. . Photoautotrophs havethe power of photosynthesis and require light. . Chemoautotrophs gain energy by oxidising inorganic chemicals such as ammonia and nitrite. Trace amountsof growth facton or vitamins Sarticularly someB viamins) are often needed. Bacteria are usually cultured in a solid medium or a liquid medium. Media are solidified with agar, (a transparent complex polysaccharide derived from red algae),which acts as a gelling agent into which the nutrients are mixed. The agar itself hasno nutritional value to the bacteria. Safety in the microbiology laboratory Rememberthat microorganisms all around- on clothes,hands,in the are air, on work surfaces, on apparatus.These microorganisms must not be inadvertently culrured during laboratory work, because they will contaminate experimentsand they may be pathogenic. Also, although the organisms commonly usedin schoolsand collegesare harmless, thereis no guaranteeof this - they may mutate into harmful forms or be contaminated with harmful forms. For these reasons aseptic techniques must always be applied rigorously to minimise risks to yourself and to colleagues. Table 1. Culture containers Petri dishes(for agar plates) Sterile plastic dishesare used to grow microbes on solid agar before disposalby autoclaving.Glass dishesmust be autoclavedbefore reuse. Bacteriolosical tubes These can hold l0 - 20 cm' of solid or liquid media. They have a loose metal cap or cofion wool bung for easyremoval and replacementwhen transferringinoculum. 1. is an agar deep-mediumallowed to solidiff in an upright position. 2. and 3. are solid agar slopes. l. molten adar Screw cappedglass bottles (McCartney bottles) These have 20 - 30 cm' capacity.They have plastic screw caps which require some dexterity when removing and replacing when transferring inoculum. solid -agar ofthe factsheet. Exam hint - microbiology exam quesfions frequenily fesf fhe candidate's knowledge about microbiologicalsafety. lt is impoftant to do microbiologypractical work to gain experience of the safetyprocedures involved.
Culture containers and instruments wire loops and wires are used for inoculating cultures.Becausethey have to be exposedto red heatthey have long metal handles(glasswould break and wood would burn). Fig 1. Inoculating instruments. loop (used for most inoculations)
sharp wire (used for culrures)
(0.5 mm diameter)
--'--> spreader(bent glass rod, used to prepare lawn plates in which bacteria form a continual 'sheet'ofcells)
(endsofspreaderscan be dipped in alcohol and then flamed, or dropped into disinfectant)
j-:: 1 I
Theseare flat glassmedicine bottlesof 100 cm' and 250 cm, capacity.They are mainly used to store large volumes of prepared sterile agar media. They may take about an hour for the agar to melt but a large number of plates can then be poured.
in Practicaltechniques microbiology
Fig 3. Pouring an agar Plate.
lacken screw top
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and 1.0,5.0 and 10.0cm'may also pasteurpipettes Sterile(autoclaved) They should have a small cotton wool plug pushed into the be required. in a mouth before autoclaving, and are preferably sterilised and stored metal container. an Ifyour school does not possess autoclavea good pressurecooker can to be used instead.You will also need a constanttemperatureincubator inoculating cabinet is also helpful (but grow the bacteria efficiently. An iot essential).This protectsthe operator from the bacteriabeing handled and minimise the risk of contaminationfrom air-borne organisms. Types of media and their preparation growth Awide variety of nutrient media capable of supporting bacterial available in a ready-prepared,dehydrated state' The are commercially then ingredients can be dissolved in water in a large beaker, and for autoclaving. Medical flats diJpensedinto suitable containersready cm' must only be partly filled, leaving an air-gap of 20-30 cm'. 10-20 dispensedinto bacteriological tubes or McCartney bottles' volumes are are The liquid media used in bacteriology are known as broths, and preparedas follows: -Dissolve . 10g meat extract, 10g of peptoneand 59 of sodium chloride the in 1dm3 of tap water. Heat gently if necessary to dissolve ingredients. .AdjustthebrothtopHT.4usingdropsoflMNaoHorlMHCl. Dispensethe broth into suitable containers(McCartney bottles)' . Autoclave at 103.4 kPa (15 psi), 121'C for 15 minutes' Ensurethat bottle caps are only lightly screwed on before autoclaving to avoid . bursting. After autoclaving and cooling?screw down the bottle caps hrmly.
3. Flame mouth of tube
4. Open lid of petri dish (as little as possible) and pour in the cooled medium 2. Allow to cool but not set
l. Melt the solid medium bY heating tubes in a water bath 7. Replacelid. Invert for storage
5. Allow to cool and set
ue, the plates should be protectedf'* atmosphericcontamination as much as possible. The plates are stored inveriedso that any condensationfallsinto the lid, not onto the agar. Inoculation and culture techniques the It is important to inoculate from young cultures' preferably in inoculum from old cultures .*pon.niiul phase of growth. If you use an growth may be Poor and atYPical. pour various types of culture may be used, for example, sffeak plates, deeps' and broth cultures' plates, .onflrr.nt plates, agar slopes' agar Bacterial cultures are usually incubatedovernight at37"C. (a) Streak plates mixed cultures into pure cultures' The This techniqueis used to separate that are formed can be used to inoculate individual bacterial colonies fresh agarplates or broth tubes.
20g of Nutrient agar is prepared in the same way as nutrient broth but dissolved into the mixture before autoclaving' After plain agai is or sterilization,when the medium is still molten, agar deeps, agarslopes agarplates can be prepared,as required' Plating out petri Stored media must be melted down prior to dispensing into sterile plate,will melt quickly, dishes.Small volumes of medium, enoughfor one melting' but 250 cm'medical flats of media will require about an hour for will contain suffrcientmedium to pour 15 to 20 plates. However,they
Fig. 2. Preparing a streak Plate
3. Flame the neck of the tube. l. Sterilise inoculating looP with red heat
2. Hold the loop in your hand and remove the plug from the tube containing the broth culture.
4. Pick up somebroth containingbacteria with the cool loop. Replacethe plug and tube in its stand.
5. Streak bacteria heavilY over the first area of the Plate.
6. Streak the bacteria from hrst to secondarea
7. Streakthe bacteria from secondto third area.
8. Streak the bacteria from third to fourth area.
8, being careful not to scmtchoJ dig up theagar surface The loopmust use the cool inoculatingloop to streakthe bacteriain actions5,6, 7 and
toiJoit""* and by besterilised redheat attoweo
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in Practicaltechniques microbiology I: Culturingbacteria
After incubation at 37oC for about 12 hours, the plate should show healy continuousgrowth cover in areasI and2,less growth cover in area3, but individual colonies in area4. The individual coloniesmay have differing due appearances to their consistingof different single species. Using a flamed, cooled inoculating loop, a single colony can be sampled and streakedout on an agarplate. When this glows it should be a pure culture of the organism. Alternatively the single colony may be inoculatedinto a broth culture for incubation and growth.
c w u,tD. u rri c u I um p ress.c o. u k
Fig. 4. Streaking a plate for pure cultures
(d) Agar slopes 'stock These are used to grow aerobicbacteriaand are useful to maintain not occupy much spaceand are easyto cultures'. This is becausethey do subcultureweekly. . Follow steps 1 to 4 describedabove under 'preparing a streakplate' to obtain a loop chargedwith inoculum. . Remove the stopper from the agar slope tube and flame the mouth of the tube. . Touch the surface of the agar with the charged loop and pull it 'wiggly'line. upwards slowly, streakingout a . Replacethe stopper,replace the rube in its stand and incubateit.
Fig 7. An agar slope.
If required,there is room to sffeak out two or three species per plate cottonwool plug -+ /,d)\ (.r)1
streak inoculatine loop
'wiggly' line of inoculum-*
-.l t: .-i --i
A broth culture can be preparedin the sameway exceptthat the inoculumis of placedinto broth instead onto an asar surface.
nutrientagar-# (b) Pour plates Each colony formed grows from a single live bacterium in the inoculum. Thus the method can be used to count the number of viable bacteriain a liquid sample. Fig. 5. Preparing a pour plate SterilePipette 1. Moltencooled mediuminoculated from a broth culture plug and 2. Replace or from a suspension roll tubebetween bacteria, containing handsto mix e.g.milk, soil and pondwater.
3. Pour the molten medium and bacteria into a sterile petri dish. Allow to set, invert and incubate.
(e) Agar deeps aerobic and These are used to grow anaerobic organismsor to Separate anaerobicorganisms.They are preparedin a similar way to the agar slope described above, except that instead of using an inoculation loop an inoculation wire is used to stab deep into the agar medium. After incubation the following types of organismmay grow'
Fig. 8. An agar deep.
aerobic bacteria micro-aerophilic bacteria (need a trace ofoxygen)
4. Exp individualbacteria colonies
(c) Confluent plates of growthis formedoverthe surface of sheet bacterial a In these, dense the medium. Fig. 6. Preparing a confluent Plate
a 2. Sterilise glassspreader by dipping the basein alcohol
and burnine it off
Anaerobic cultures Any of the culture techniques described above can be used to gtow anaerobic organisms,but the cultures must be gfown in the absenceof In oxygen, particularly in the caseof obligate anaerobes. theseorganisms growth will be inhibited by the presenceof oxygen. The culture plates (or bottles) can be kept in anaerobicjars. These are evacuated of air (containing oxygen) and the air is replaced by an usually consistingof 80% nitrogen, l0% carbon dioxide and atmosphere by l0% hydrogen.Thesegasesare generated packetsof chemicalsplaced in the jar before sealing and clamping the lid. Initially the lid valves are open to enable the generatedgases to push out the air (oxygen). When this has happenedthe valves are closed. An alternative way of providing an anaerobic atmosphere is to vent nitrogen from a gas cylinder in through the open valves, closing the valves when air is evacuated. Anaerobicjars are availableto contain9,12 or 48 petri dishes. Exam Hint - exam guesflons are often sef fo fest fhe practical experience of candidates. You may be asked to describe media preparation or inoculation and culture techniques.
Practicaltechniques microbiology I: Culturing bacteria in
Practice Questions (a) Distinguish'nutrientbroth'from'nutrient agar' l. 2 (b) What are the essentialbasic contentsof any bacteriological nutrient media? 6 (c) What are the main aims when growing bacteriaon a streaknlate]
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What is the minimum recommendedpressure,temperature and time for sterilisationin an autoclave? 3 ( e ) D i s i n f e c t a n t sm u s t b e a p p l i e d ' s t r o n g e n o u g h f o r l o n g enough'. Suggest why. 3 2. (a) Describe how you would obtain pure broth cultures of the two aerobicorganisms, Escherischiacoli md Staphvlococcus aureus from a mixed broth culture of the two organisms. 5 Outline how you would prepare pure plate cultures of a soil water mixture of obligate anaerobicbacteria speciesobtained from waterloggedclay soil. 4 Suggesthow you can reduce the cross-infection risks involved when red-heatflaming inoculation loops. 3 Why are agar plates and plate cultures incubated and stored upside down? 2 What do you understand by the terms 'stock culture' and 'subculfuring'? 3
they touch anything else; 3 becausecondensation may occur (from water in the agar); it is safestif the drops of water collect in the lid rather than on the bacterialgrowth; 2 a 'stock culture'is a culnre that is maintainedto provide a supply of a particular bacterial species/microorganism over a long period of time; it is usually an agar slope culture and needsregular subculturing for continuity; 'subculturing' is the term used to describe the inoculation of fresh medium usins bacteria/microorsanism from an old culfure, 3
(a) (b) (c)
Answers (a) nutrient broth remains in liquid form, nutrient agar sets as a l. gel/is solid; bacteria grow in nutrient broth but usually on the surface of nutrient agar: 2 (b) a source of carbon/glucose/simple sugar/saltof organic acid; a sourceof nitrogenipeptides/peptone/amino acids/ammoniumsalts; mineral salts/anytwo correct cationsand any two correct anions stated:water; energy source; vitamins/growttr factors/ B-vitamins; 6 (c) to separatetwo or more species of bacteria from mixed culfurelsource; sfteakingtechniquereducesthe denslryofbacterial growth resulting in isolatedcolonies; thesecan then be subcultured/harvested grown in pure culturg and
(d) 15 psi/103.4 kPa; 121'C; 15 minutes; 3 (e) not all bacteriawill be equally susceptibleto the disinfectant; thus it must be strong enough to kill/inhibit the most resistant individuals; and be allowed to act long enough to reduce the bacterial populationto a safelevel;; 3 2. (a) preparea sfteakplate from an inoculum of the mixed broth culture; correct details of how this is done;; (2 marks - details of inoculation /streaking technique/incubation) use isolatedS. aureuscoloniesas inoculum for a broth culture and isolatedE. coli coloniesas inoculum for the other broth culture: some conect practical detail of inoculating/incubatingiaseptic technique involved in producing a broth culture; 5 ref to the need to grow all cultures in anaerobicjars/in the absenceof oxygen; preparea broth culture using the soil water mixture as inoculum (to increasethe population density of the bacteria); preparea streakplate from an inoculum of this broth culture; subculturedistinct isolated colonies onto sterile agar plates and incubate anaerobicallv: never overload inoculation loop becauseresidual material could be dispersedas an aerosol(particularly when nearing the flame); always draw the loop through the flame from behind so that the flame is betweenyou and the bacteria/anyformed aerosol; work under a safetyhood/do not allow other people near to the flame when flaming/flame contaminatedloops promptly before
Appendix 1. Always wear a laboratory coat when working in the laboratory. This will protect your clothes from contamination with the cultures you handleand will protectyour clothesfrom splasheswhich may occur. Laboratorycoats shouldnot be takenout of the laboratorybecauseof the risk of spreading (unlessin a sealedlaundrybag). contamination 2. Coverany minor cuts on exposedparts of your body with a surgical plasterbeforeworkingwith microbes. 3. Always wipe down the top of your bench with disinfectant the at beginning period.All used cotton-wool and end of each laboratory or papermust be discarded burning. absorbent for 4. Hand-to-mouth operationssuch as eating, smoking and licking of labelsare forbidden. Dampenstickylabelswith water,(not with your tongue),or mark containers with a felt pen or wax pencil. 5. Never pipette cultures by mouth. Always use a teat pipette for transferring smallvolumesof liquidcultures. not forcefully Do squeeze the liquidfrom the pipettebecausethis can producean aerosol. 6. lf a culture is spilt tell your supervisor once so that appropriate at remedialaction can be taken. lf an accidentoccurs,alwaysreportit to your supervisor. 7. When transferring properlysterilisethe inoculating micro-organisms, instrument beforeand after the transfer. The inoculating wire should be raisedto red heat on each occasion.Be carefulwhen loops are contaminatedwith inoculum because splattering and aerosol formation can occur.(lt is safest, the operator, bringthe loop into for to the flame on the side furthestfrom the operator- move the loop throughthe flametowardsthe operator). 8. The openingof any containercontaininga culturecan produce an aerosol.Only open containers necessary. if Never sniff the cultures unlessyou are told that it is safe to do so. 9. Neverremovecultures from the laboratory. 10.Usedcultures,petridishesor otherglasswareshouldbe autoclaved after the experiment.Microscopeslides with live mounts must be immersedin disinfectant immediately after use. ll.Thoroughly wash and dry your hands before leavingthe laboratory. Hot water,soap and disposable towelsmust be available. All used culturesmust be disposedof immediately, becausethey are a hazard to other workersand may contaminateother cultures: 1. Plasticpetridishescontaining agar culturesmust be autoclaved, a in small metal bucketor in an autoclave bag and then disposedof via the refusebin. 2. Culturesgrown in glassware must be autoclavedand the glassware then washed for further use. After autoclaving,the warm liquid mediumcan be flushedaway down a sink with hot water. 3. lmmerse used microscopeslides in a beaker containingfreshly prepared2% Chloros. 4. lmmediately place used pipettes in jars with freshly prepared 2o/o Chloros. Wash and sterilise pipetteslater. the 5. Treatspillages cultures clothing of on with a non-bleaching, non-staining disinfectant, example1% Cetavlon for 6. Spillages culturesare best dealt with by swabbingthe contaminated of areawitha strongsporicidal disinfectant, example,10%Chloros. for (Lysol is not recommended it is not sporicidal, it is also a stronginitant). as and
Acknowledgements This Factsheet was researched and written by Martin Grffin. Curriculum Press. Unit 3058, The Big Peg, 120 VyseStreet, Birmingham 818 6NF Bio Factsheets mav be copiedfree ofcharge by teaching stalfor students, provided that their school is a registered subscriber. No part oJ'theseFactsheets ma\, be reproduced, stored in a retrieval system, or transmitted, in anv- other form or by any other means, without the prior permission oJ the publisher 1,SSN1351-5136
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