Quantitative Analysis of Microbial Populations through Standard Viable Plate Count Methods
Damiles, Johmar, De Vera, Alyssa Gail, Elio, Charlene Lorraine, Fernandez, Gino Miguel*
2BIOLOGY6, Department of Biology, College of Science, UST, Manila
*
[email protected]
Abstract Microorganisms are within nature, they occur and are studied within the liing enironment or inside a la!oratory, they are not isolated then studied as di""erent and se#arate organisms, !ut as a whole, as #o#ulations within nature, either in colonies or as dis#ersed units sus#ended in a medium or in a sus#ended animation$ %our #late and &#read %late method are the two most common methods used in the isolation o" microorganisms "rom their natural ha!itat$ Additionally, Additionally, these two #rocesses gie the most a##ro'imate or estimate num!er o" organisms #resent in a #o#ulation o" microorganisms$ Keywords: &#read %late Method, %our %late Method, (solation, &us#ended in a Li)uid Medium
Introduction Microorganisms are within nature, they occur and are studied within the liing enironment or inside inside a la!ora la!orator tory, y, they are are not isolat isolated ed then then studied as di""erent and se#arate organisms, !ut as a whole, as #o#ulations within nature, either in coloni colonies es or as dis#er dis#ersed sed units units sus#en sus#ended ded in a medium or in a sus#ended animation$ %our #late and and &#re &#read ad %lat %late e meth method od are are the two two most most comm common on metho ethods ds used used in the isol isolat atiion o" micr microo oorg rgani anism sms s "rom "rom thei theirr natu natura rall ha!i ha!ita tat$ t$ Additionally, Additionally, these two #rocesses gie the most a##ro' a##ro'im imate ate or estim estimate ate num!er num!er o" organi organisms sms #resent #resent in a #o#ulatio #o#ulation n o" microorga microorganisms nisms$$ he #our #late techni)ue can !e used to determine the num!er num!er o" micro! micro!es+ es+mL mL or micro! micro!es+ es+gra gram m in a s#ecimen$ (t has the adantage o" not re)uiring #reiously #re#ared #lates, and is o"ten used to assay !acterial contamination o" "oodstu""s$ he #rinci#le ste#s are to - #re#are+dilute the sam#le .- #lace an ali)uot o" the diluted sam#le in an em#ty sterile #late /- #our in 0 mL o" melted agar which has !een cooled to 10o C, swirl to mi' well 1- let cool undistur!ed to solidi"y on a "lat ta!le to# 0- inert and incu!ate to deelo# colonies$ Each Each colony re#resents a 2colony "orming unit2 3CF4-$ For o#timum accuracy o" a count, the
#re"erred range "or total CF4+#late is !etween /5 to /55 colonies+#late colonies+#late$$ 6ne disadant disadantage age o" #our #late #lates s is that that em!edd em!edded ed coloni colonies es will will !e much much smaller than those which ha##en to !e on the sur"ac sur"ace, e, and must must !e care"u care"ully lly scored scored so that that none are oerloo7ed$ Also, o!ligate aero!es may grow #oorly i" dee#ly em!edded in the agar$ 6n the othe otherr hand hand,, s#rea #read d #lat #late e metho ethod d can can determine the num!er o" !acteria in a solution that can !e readily )uanti"ied$ (n this techni)ue, the sam#le is a##ro#riately diluted and a small ali)uot trans"erred to an agar #late$ he !acteria are then distri!uted eenly oer the sur"ace !y a s#ecial strea7 strea7ing ing techni techni)ue )ue$$ A"ter A"ter coloni colonies es are grown, grown, they are counted and the num!er o" !acteria in the original original sam#le sam#le calculated$ calculated$ he end #oint o" our analysis is the num!er o" colony "orming units #er #er mL 3CF4 3CF4+m +mLL- sinc since e we are are coun counti ting ng the the num!er o" colonies rather than the actual num!er o" !acteria$ 8e are assuming that the each ia!le !acteria !acteria in the sus#ension sus#ension will "orm an indiidual indiidual colony, which is a alid assum#tion i" we do all the techni)ues #ro#erly$ CF4+mL is actually a more use"ul determination than counting all the !acteria under a microsco# microsco#e e !ecause !ecause in many !acterial !acterial #o#ulations some signi"icant num!er will !e dead cells and thus o" no interest$
Methodology
Pour Plate Technique
he researchers #re#ared / test tu!es o" diluent containing 9$5 ml each o" $5: #e#tone !roth or 5$;0: saline 3
., 5>/, and 5>1$ 4sing a ru!!er !ul! as#irator, the researchers ase#tically #i#et $5 ml "rom a .1 hour old culture !roth into the "irst dilution !lan7$ he contents o" the "irst dilution !an7 were then mi'ed !y rotating the tu!e !etween the #alms o" the researcher=s hands$ he used #i#ettes were then discarded or di##ed in a !ea7er o" 5$0: !leach disin"ectant$ A second $5>ml serological #i#ette was used to trans"er $5 ml "rom the "irst dilution !an7 to the second one$ he contents were mi'ed once again$ he same #i#ette was used to trans"er the contents o" the "irst dilution !an7 to two #lates mar7ed as the same with the "irst dilution !an7$ he #i#ette was then discarded in the disin"ectant$ A third serological #i#ette was then used to trans"er the contents o" the second dilution !an7 to the third$ he same serological was used to de#osit the contents o" the second dilution !an7 to two #lates la!eled the same as the second dilution !an7$ Discard the #i#ette in the disin"ectant$ 0>.5 ml o" molten, sterile %CA was #oured ase#tically into each o" the #lates$ he #lates were then gently swirled !y touching the #late with the researcher=s #ointing and middle "inger and moing it !ac7 and "orth, right>le"t direction to mi'$ he agar was allowed to harden$ he #lates were then wra##ed in news#a#er and enclosed in #lastic !ags "or incu!ation "or .1 hours at room tem#erature$ A"ter incu!ation, the colonies in the #late were then counted with /5> /55 colonies using a #ermanent mar7er to #reent counting o" a colony twice$ he aerage num!er o" colonies was ta7en$ he aerage num!er o" colony "orming units #er ml was ta7en$ Spread Plate Technique
A 05 ml %CA sam#le was #re#ared according to instructing and was sterilized ia the autoclae machine$ he sterilized sam#le was then ta7en out o" the autoclae machine and was allowed to cool "or a !it$ (t was then #oured indiidually into ? sterile #etri dishes and was allowed to solidi"y$ hese %CA #lates were la!eled accordingly$ A 5 "old serial dilution o" the .1 hour !roth culture was then #re#ared li7ewise with the #our #late method$ 6nce again, the researchers
had / test tu!es containing 9$5 ml o" sterile 5$;0: saline$ A $5 ml o" the .1 hour !roth culture was ase#tically withdrawn and introduced to the "irst test tu!e that was la!eled accordingly to the assigned dilutions to each o" the researchers$ he content was then thoroughly mi'ed$ A sterile $5 ml serological #i#ette was used to #i#ette out $5 ml "rom the "irst test tu!e and then introduced to the second test tu!e la!eled accordingly to assigned dilution to the res#ectie researchers$ he same serological #i#ette was then used to introduce 5$ ml "rom the "irst test tu!e to two o" the %CA #lates that was #reiously #re#ared and la!eled accordingly$ he 5$ ml was dro##ed in the center o" the %CA #lates$ 4nli7e with the #our #late method, only 5$ ml was introduced to the #lates$ he used serological #i#ette was then disin"ected in a !ea7er with 5$0: !leach or disin"ectant$ Another serological #i#ette was o!tained and $5 ml was withdrawn "rom the second test tu!e and was added to the third test tu!e la!eled accordingly$ he content o" the third test tu!e was then mi'ed$ he same serological #i#ette was used to withdraw 5$ ml "rom the second test tu!e and introduced to two %CA #lates$ hen, another 5$ ml serological #i#ette was o!tained, this was used to #i#ette out 5$ ml "rom the third test tu!e and was then introduced to the last two #etri dishes$ An L>sha#ed glass rod was di##ed in 90: ethanol in a !ea7er and withdrawn$ he L>sha#ed rod was then "lamed oer$ he "lame was le"t to !urn and disa##ear$ A %CA #late was then o!tained and the L>sha#ed rod was used to s#read the #reiously dro##ed sam#le all oer the %CA #late$ his #rocess was re#eated "or all %CA dishes$ he #lates were then wra##ed in a news#a#er, and enclosed in #lastic !ags$ hese were then incu!ated "or .1 hours$ he CF4+ml "ormula was then com#uted, similar with the #our #late method, !ut the olume #lated that is su!stituted in the "ormula should !e 5$ ml$
esults and !iscussion Pour Plate Technique
he inerted #late cultures were ta7en out a"ter incu!ation "or at least .1 hours$ he #lates e'hi!ited #ro#er distinction #rior to the dilution o" the sam#le$ Colonies no matter how small has to !e counted as they are considered to "orm larger and !igger colonies as it ages$ he least diluted #late 5>? had the most num!er o" colonies "ormed, to the #oint that one #late e'ceeded more
than /55 colonies and was assessed with @too many to count, een the dilution o" 5 >? had one trial assessed with too many to count$ he most diluted sam#les on the other hand, had a num!er that would !e #ro#ortional to its dilution$ Due to the agar !eing de#osited a"ter the sam#le has !een #laced in the #late, instead o" colonies haing to settle only on the sur"ace o" the agar, there were some which settled in the middle o" the agar medium, thus, ma7ing it more di""icult to count the colonies unli7e the s#read #late techni)ue which had its colonies settle only on the sur"ace o" the medium$ his techni)ue may also hae 7illed or stunted the growth o" o!ligate aero!ic microorganisms !ecause o" their settlement in the middle o" the medium$ he three #lates with reasona!le colony count was aeraged !y mathematical calculations and resulted to a concentration o" 1$5B'5; cells #er mL and was a reasona!le "or sewage water sam#le$
organisms$
Spread Plate Technique
he inerted #late cultures were ta7en out a"ter incu!ation "or at least .1 hours$ he #lates e'hi!ited #ro#er distinction #rior to the dilution o" the sam#le$ Colonies no matter how small has to !e counted as they are considered to "orm larger and !igger colonies as it ages$ he least diluted #late, 5>. had the most num!er o" colonies$ ecause, s#read #late method was used, the colonies were almost eenly s#read throughout the #late and ma7ing it easier to count$ he colonies in this method were settled only on to# o" the solidi"ied agar$ he three #lates with their res#ectie colony counts was aeraged and resulted to a concentration o" 0$B.'5 >. cells #er ml and this was the result "or the researcher=s grou# "or #ond water$
Conclusion From the e'#eriment, the researchers hae in"erred that the !est way o" )uantitatie analysis in micro!ial #o#ulations would !e the s#read #late techni)ue$ &#read #late techni)ue is a !etter way o" determining the amount o" colonies in the sam#le !ecause it does not damage the colonies in any way, and the colonies would !e almost eenly distri!uted through the #late thus ma7ing it easier to count$ %our #late could 7ill some o" the microorganisms !ecause the researchers would hae to #our molten agar into their diluted sam#le, and the heat might hae 7illed o"" some o" the
eferences
htt#++www.$hendri'$edu+!iology+Cell8e!+echni)ues+micros#read$htm
. htt#++!iology$clc$uc$edu+"an7hauser+La!s+Micro!iology+MeatMil7+%our%late$html / La!oratory Manual in General Microbiology. 3.51-$ Manila 4niersity o" &anto omas$
