Direct microscope method

Direct microscope method

1. Bacterial Cells Enumeration
DIRECT MICROSCOPIC METHOD (TOTAL CELL COUNT)
Yousef Elshrek

2. •DIRECT MICROSCOPIC METHOD (TOTAL CELL COUNT)
•In the direct microscopic count, a counting chamber consisting of
a ruled slide and a coverslip is employed.
•It is constructed in such a manner that the coverslip, slide, and
ruled lines delimit a known volume.
•The number of bacteria in a small known volume is directly
counted microscopically and the number of bacteria in the larger
original sample is determined by extrapolation.

3. •ThePetroff-Haussercountingchamber(seeFig.1AandFig.1B)forexample,has
smalletchedsquares1/20ofamillimeter(mm)by1/20ofammandis1/50ofa
mmdeep.
http://faculty.ccbcmd.edu/courses/bio141/labmanua/lab4/images/abstrans.jpg
•Fig.(1A).LargeDouble-LinedSquareofa
Petroff-HausserCounter.Thelarge,double-
linedsquareholdsavolumeof1/1,250,000of
acubiccentimeter.Usingamicroscope,the
bacteriainthelargesquarearecounted.
•Fig.(1B)Petroff-HausserCounterasseen
throughaMicroscopeThedouble-lined
"square"holding1/1,250,000ccisshownby
thebracket.Thearrowshowsabacterium.
http://faculty.ccbcmd.edu/courses/bio141/labmanua/lab4/images/abstrans.jpg

4. •The volume of one small square therefore is 1/20,000 of a cubic mm or
1/20,000,000 of a cubic centimeter (cc).
•There are 16 small squares in the large double-lined squares that are counted,
making the volume of a large double-lined square 1/1,250,000 cc.
•The normal procedure is to count the number of bacteria in five large double-
lined squares and divide by five to get the average number of bacteria per large
square.
•This number is then multiplied by 1,250,000 since the square holds a volume of
1/1,250,000 cc, to find the total number of organisms per cc in the original
sample. If the bacteria are diluted, such as by mixing the bacteria with dye
before being placed in the counting chamber, then this dilution must also be
considered in the final calculations.

5. •Theformulausedforthedirectmicroscopiccountis:
•If the bacteria are diluted, such as by mixing the bacteria with dye before
being placed in the counting chamber, then this dilution must also be
considered in the final calculations

6. •Structure of Petroff-Hausser
Counting Chamber
•DirectMicroscopicMethod(TotalCell
Count)
1.Rulingarethecenterlinesofthe
groupsofthree.(Theseareindicated
intheillustrationFig2).
2.Thecentralsquaremillimeterisruled
into25groupsof16smallsquares,
eachgroupseparatedbytriplelines,
themiddleoneofwhichisthe
boundary(seeFig.108C).
3.Theruledsurfaceis0.02mmbelowthe
coverglass,sothatthevolumeovera
squaremillimeteris0.02cubicmm.
http://hausserscientific.com/products/images/neubauer_ruling.gif
Fig. (2)Petroff-Hausser Counting Chamber

7. 4.Count all cells within this center square millimeter (1mm x 1mm area).
Fig. (3)Petroff-Hausser Counting Chamber in detail.
•Theruledareaofthehemocytometer
consistsofseverallarge1x1mm
(1mm²)squares
•Whicharesubdividedinthreeways;
0.25x0.25mm(0.0625mm²),0.25x
0.20mm(0.05mm²)and0.20x0.20mm
(0.04mm²).
•Thecentral,0.20x0.20mmmarked;1x
1mmsquareisfurthersubdividedinto
0.05x0.05mm(0.0025mm²)squares.
•Holdthecoverslip(0.1mm)atthe
raisededgesofhemocytometer,which
giveseachsquareadefinedvolume.

8. Area Volume at 0.1mm depth
1 x 1 mm (red) 1 mm
2
100 nl
0.25 x 0.25 mm (1/16) (green) 0.0625 mm
2
6.25 nl
0.25 x 0.20 mm (1/20) 0.05 mm
2
O.5 nl
0.20 x 0.20 mm (1/25) (Yellow) 0.04 mm
2
0.4 nl
0.05 x 0.05 mm (1/400) (blue) 0.0025 mm
2
0.25 nl
Table (10) shows area and depth volume of hemocytometer
https://3.bp.blogspot.com/QPDc7ZXFQOU/WdyfPeCBgTI/AAAAAAAAA0A/JNylMZ9G
dIUlzEfI8unIUr6ONhWSxrRwACLcBGAs/s400/hemeo2%2Bkar%25281%2529.jpg
Fig. (4) Area of each square in Hemocytometer
Red square = 1mm
2
Green square = 0.0625 mm
2
Yellow square = 0.04 mm
2
Blue square = 0.0025 mm
2

9. Fig. (5)
https://163602-560839-raikfcquaxqncofqfm.stackpathdns.com/wp-content/uploads/2019/02/Direct-Microscopic-Counts.jpg

10. •MATERIAL REQUIRED
1.Culture: 18-24-hour nutrient broth of E. coli
2.Sample: Food sample
3.Reagents: Methylene blue stain 37 Direct Microscopic Examination of Food
4.Equipment and glassware:
a.Petroff-Hausser Counter (one for each blended food sample (10
-1
dilution) or each
given culture)
b.Pipettes with pipette aids
c.blender, or stomacher (for food sample)
d.Microscope
e.test tubes

11. •PROCEDURE
1.Pipette1.0mlofthesampleofE.coliinto
atubecontaining1.0mlofthedye
methyleneblue.Thisgivesa1/2dilution
ofthesample.
2.UsingaPasteurpipette,fillthechamberof
aPetroff-Haussercountingchamberwith
this1/2dilution.
3.Placeacoverslipoverthechamberand
focusonthesquaresusing400X(40X
objective).
4.Countthenumberofbacteriain5large
double-linedsquares.Forthoseorganisms
onthelines,countthoseontheleftand
upperlinesbutnotthoseontherightand
lowerlines.Dividethistotalnumberby5
tofindtheaveragenumberofbacteriaper
largesquare.
http://www.microbehunter.com/wp/wp-content/uploads/2010/06/counting_chamber6.jpg
Fig. (6) Counting chamber seen from the side.
5.Calculatethenumberofbacteriaperccasfollows:
•Thenumberofbacteriapercc=theaverage
numberofbacteriaperlargesquare×thedilution
factorofthelargesquare(1,250,000)×thedilution
factorofanydilutionsmadepriortoplacingthe
sampleinthecountingchamber,suchasmixingit
withdye(2inthiscase).
•In case of DMC of milk sample, use Breed’s slide and
Breed’s pipette

12. •OBSERVATIONS
•To determine the concentration of bacteria in the
original culture use the following formula:
•Formula for the counting chamber
•Use this formula for calculating the number of cells per
ml from the count obtained using a counting chamber.
1.Nc is the average number of cells counted per square
2.D is the dilution of the samples placed on the slide.
For example:
The 10
3
is there as a conversion
factor from mm
3
as measured by the
chamber to cm
3
(or ml) as typically
expressed for culture density. Here is a
more detailed explanation of that
conversion factor:
1 ml = 1 cm
3
= 1 cm ×1 cm ×1 cm
1 cm = 10 mm
so, 1 ml = 10 mm ×10 mm ×10 mm
or 1 ml = 10
3
mm
3

13. •RESULTS
•Theycanbecalculatedusingthefollowingexample:
•Ifyouuseonedrop(withoutdilution)fromabrothculture:andfindan
averageof2.31cellspersquares,yourresultswouldbe:
•Calculationofcellnumberfromacountingchamber
•Ifanaverageof2.31cellsiffoundina10
-1
dilution,theformulawould
appearasshownherewitharesultof4.62x10
8
cellspermlofculture.

14. •PRECAUTIONS
1.Usingacapillarypipette,placeadropofthebrothcultureattheedgeofthecover
slipalways.Capillaryactionwilldrawtheliquidunderthecoverslip.Then,wait
for1-2minutesforthemovementtostopandthecellstosettle.
2.Alwaysfocusthelow-powerobjectiveonthegridinthecenteroftheslide;you
shouldseeacrosshatchedareacontaining25squareseachcontaining16smaller
squares.
3.Donotusetheoilimmersionlenswiththesechamber
4.Alwayscountthenumberofbacteriain10-15ofthe1/400mm
2
squaresand
calculateanaveragecellnumber.
5.Useadilutionsuchthattherearenomorethanthreecellsineachsmall(1/400
mm
2
)squareandthetotalnumberofcellscountedisatleast100,tobe
statisticallycorrect.

15. •Second Procedure
•Counting cells in a Petroff Hauser
•Procedure
1.A Petroff Hauser is a device that is used for counting cells. It is a modified microscope slide,
containing two identical wells, or chambers, into which a small volume of a cell suspension is
pipetted by removing 100 µL of cell suspension and placed it in a micro-centrifuge tube.
2.Dilute the suspension by adding 100 µL of Trypan blue. Trypan blue is a dye that helps us
distinguish between living and dead cells. The dye passes through the membranes of dead
cells so they will appear blue under a microscope.
3.Living cells exclude and will appear mostly clear. Load both chambers by pipetting the
suspension under the cover slip.
4.Place the hemocytometer under the microscope. Each chamber is divided into a grid pattern,
consisting of 9 large squares. Each square has the same dimensions and contains 10 to the
negative-fourth power mL of suspension.

16. 5.The rules for counting cells
sometimes differ from lab-to-lab. In
this lab experiment, counting cells in
the 4 large corner squares and the
center square.

17. 6.There are 10 viable cells and 1 non-viable
cell (blue color) in the first square
Countonlythecellsthattouchinsideboundaries
andignorethecellsthatoutsidetouchoutthe
boundaries,youneedtocountthenumberofboth
livinganddeadcells,remember,thedeadcells
willappearblue,occasionallyyouwillsee
artifacts-objectsordebristhatappearblurryand
don'thaveawell-definedshape,thisisanexample
ofanartifact,youwon'tincludeitinourcount,
properstorage,cleaning,andhandlingofthe
hemocytometerwillminimizethenumberof
artifacts.

18. http://www.microbehunter.com/wp/wpcontent/uploads/2010/06/counting_chamber5.jpg
Countonlythecellsthattouchinside
boundariesandignorethecellsthat
outsidetouchouttheboundaries,youneed
tocountthenumberofbothlivingand
deadcells,remember,thedeadcellswill
appearblue,occasionallyyouwillsee
artifacts-objectsordebristhatappear
blurryanddon'thaveawell-defined
shape,thisisanexampleofanartifact,
youwon'tincludeitinourcount,proper
storage,cleaning,andhandlingofthe
hemocytometerwillminimizethenumber
ofartifacts.

19. 7.There are 9 viable cells
and no non-viable cells
in the top-right square

20. 8.Next let us count the bottom-
right square, there are 11
viable cells and no non-
viable cells.

21. •Thereare10viablecells
and2non-viablecellsinthe
leftbottomsquare
finally,countthecellsinthecentersquare,
Sometimescellswillappearasclumpsorsmall
groups,itmaybedifficulttodetermineexactly
howmanycellsareinagroup,themethodof
countingclumpsofcellsdiffersfromlabtolab,
sobesuretofollowtheprocedureinyourlab,in
thislabyouwillcountthisclumpas2cells.

22. 10.There are 14 viable cells
and no non-viable cells in
the center square.

23. 11.The total number of viable or
living cells from all 5 squares is
54, and non-viable cells is 3.

24. •Calculations
•Total viable cells: 54
•Total nonviable cells: 03
1.% of viable cells: 94.7%
2.% of nonviable cells: 05.3%
3.Average number of cells square =10.8
4.Dilution factor =2
5.Concentration (viable cells/ml) = 2.16x10
5
cells /ml

26. •N.B. How to calculate the dilution factor.
1.The dilution equals the final volume divided by the volume of cells.
2.Th final volume is 200 µL, because you started with 100 µL of cells and
added another 100 µL of trypan blue.
3.200 divided by 100 is 2. Therefore, the dilution factor is 2.
•Cell Viability Testing with Trypan Blue Exclusion Method
•The Trypan Blue dye exclusion test is used to determine the number of viable
cells present in a cell suspension. It is based on the principle that live cells
possess intact cell membranes that exclude certain dyes, such as trypan blue,
Eosin, or propidium, whereas dead cells do not. When a cell suspension is simply
mixed with the dye and then visually examined to determine whether cells take
up or exclude dye. A viable cell will have a clear cytoplasm whereas a nonviable
cell will have a blue cytoplasm.
•Periodic cell viability assessment provides an early indicator of the quality of
your fresh cells prior to freezing. Viabilities of greater than or equal to 95% are
excellent.

27. •Advantages of Direct Microscopic Count
•Rapid, Simple, and easy method requiring minimum equipment. Morphology
of the bacteria can be observed as they counted.
•Very dense suspensions can be counted if they are diluted appropriately.
•Limitations of Direct Microscopic Count
•Although rapid, a direct count has the disadvantages that both living and dead
cells are counted.
•Only dense suspensions can be counted (>107 cells per ml), but samples can be
concentrated by centrifugation or filtration to increase sensitivity.
•It is not sensitive to populations of fewer than 1 million cells.
•Small cells are difficult to see under the microscope, and some cells are
probably missed.
•Precision is difficult to achieve
•A phase contrast microscope is required when the sample is not stained.

28. •References
1.Cappuccino, J. and Welsh, C. (2014).Microbiology: A Laboratory
Manual, Global Edition. 1st ed. Pearson Education
2.Sastry A.S. & Bhat S.K. (2016). Essentials of Medical Microbiology.
NewDelhi :Jaypee Brothers Medical Publishers.
3.http://textbookofbacteriology.net/growth_2.html
4.http://repository.uobabylon.edu.iq/mirror/resources/paper_2_13669_7
49.pdf
5.https://courses.lumenlearning.com/boundless-
microbiology/chapter/counting-bacteria/
6.http://ecoursesonline.iasri.res.in/md/resource/view.php?id=101513
7.https://youtu.be/pP0xERLUhyc