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. • The Petroff-Hausser counting chamber (see Fig. 1 A and Fig. 1B) for example, has
small etched squares 1/20 of a millimeter (mm) by 1/20 of a mm and is 1/50 of a
mm deep.
http://faculty.ccbcmd.edu/courses/bio141/labmanua/lab4/images/abstrans.jpg
• Fig. (1A). Large Double-Lined Square of a
Petroff-Hausser Counter. The large, double-
lined square holds a volume of 1/1,250,000 of
a cubic centimeter. Using a microscope, the
bacteria in the large square are counted.
• Fig.(1B) Petroff-Hausser Counter as seen
through a Microscope The double-lined
"square" holding 1/1,250,000 cc is shown by
the bracket. The arrow shows a bacterium.
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. • The formula used for the direct microscopic count is:
• 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
• Direct Microscopic Method (Total Cell
Count)
1. Ruling are the center lines of the
groups of three. (These are indicated
in the illustration Fig 2).
2. The central square millimeter is ruled
into 25 groups of 16 small squares,
each group separated by triple lines,
the middle one of which is the
boundary (see Fig. 108C).
3. The ruled surface is 0.02 mm below the
cover glass, so that the volume over a
square millimeter is 0.02 cubic mm.
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.
• The ruled area of the hemocytometer
consists of several large 1 x 1 mm
(1mm²) squares
• Which are subdivided in three ways;
0.25 x 0.25 mm (0.0625 mm²), 0.25 x
0.20 mm (0.05 mm²) and 0.20 x 0.20 mm
(0.04 mm²).
• The central, 0.20 x 0.20 mm marked; 1 x
1 mm square is further subdivided into
0.05 x 0.05 mm (0.0025 mm²) squares.
• Hold the cover slip (0.1 mm) at the
raised edges of hemocytometer, which
gives each square a defined volume.

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 = 1mm2
Green square = 0.0625 mm2
Yellow square = 0.04 mm2
Blue square = 0.0025 mm2

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. Pipette 1.0 ml of the sample of E. coli into
a tube containing 1.0 ml of the dye
methylene blue. This gives a 1/2 dilution
of the sample.
2. Using a Pasteur pipette, fill the chamber of
a Petroff-Hausser counting chamber with
this 1/2 dilution.
3. Place a cover slip over the chamber and
focus on the squares using 400X (40X
objective).
4. Count the number of bacteria in 5 large
double-lined squares. For those organisms
on the lines, count those on the left and
upper lines but not those on the right and
lower lines. Divide this total number by 5
to find the average number of bacteria per
large square.
http://www.microbehunter.com/wp/wp-content/uploads/2010/06/counting_chamber6.jpg
Fig. (6) Counting chamber seen from the side.
5. Calculate the number of bacteria per cc as follows:
• The number of bacteria per cc = the average
number of bacteria per large square × the dilution
factor of the large square (1,250,000) × the dilution
factor of any dilutions made prior to placing the
sample in the counting chamber, such as mixing it
with dye (2 in this case).
• 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 103 is there as a conversion
factor from mm3 as measured by the
chamber to cm3 (or ml) as typically
expressed for culture density. Here is a
more detailed explanation of that
conversion factor:
1 ml = 1 cm3 = 1 cm × 1 cm ×1 cm
1 cm = 10 mm
so, 1 ml = 10 mm × 10 mm × 10 mm
or 1 ml = 103mm3

13. • RESULTS
• They can be calculated using the following example:
• If you use one drop (without dilution) from a broth culture: and find an
average of 2.31 cells per squares, your results would be:
• Calculation of cell number from a counting chamber
• If an average of 2.31 cells if found in a 10-1 dilution, the formula would
appear as shown here with a result of 4.62 x 108 cells per ml of culture.

14. • PRECAUTIONS
1. Using a capillary pipette, place a drop of the broth culture at the edge of the cover
slip always. Capillary action will draw the liquid under the cover slip. Then, wait
for 1-2 minutes for the movement to stop and the cells to settle.
2. Always focus the low-power objective on the grid in the center of the slide; you
should see a cross hatched area containing 25 squares each containing 16 smaller
squares.
3. Do not use the oil immersion lens with these chamber
4. Always count the number of bacteria in 10-15 of the 1/400 mm2 squares and
calculate an average cell number.
5. Use a dilution such that there are no more than three cells in each small (1/400
mm2) square and the total number of cells counted is at least 100, to be
statistically correct.

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
Count only the cells that touch inside boundaries
and ignore the cells that outside touch out the
boundaries, you need to count the number of both
living and dead cells, remember, the dead cells
will appear blue, occasionally you will see
artifacts - objects or debris that appear blurry and
don't have a well-defined shape, this is an example
of an artifact, you won't include it in our count,
proper storage, cleaning, and handling of the
hemocytometer will minimize the number of
artifacts.

18. http://www.microbehunter.com/wp/wpcontent/uploads/2010/06/counting_chamber5.jpg
Count only the cells that touch inside
boundaries and ignore the cells that
outside touch out the boundaries, you need
to count the number of both living and
dead cells, remember, the dead cells will
appear blue, occasionally you will see
artifacts - objects or debris that appear
blurry and don't have a well-defined
shape, this is an example of an artifact,
you won't include it in our count, proper
storage, cleaning, and handling of the
hemocytometer will minimize the number
of artifacts.

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. • There are 10 viable cells
and 2 non-viable cells in the
left bottom square
finally, count the cells in the center square,
Sometimes cells will appear as clumps or small
groups, it may be difficult to determine exactly
how many cells are in a group, the method of
counting clumps of cells differs from lab to lab,
so be sure to follow the procedure in your lab, in
this lab you will count this clump as 2 cells.

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) = 2.16x105 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.
New Delhi : 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