Lab 2: Measurement and counting of cells using microscope

Name of group member:
1. Chok Wen Xin (133291)2. Leong Kah Yan (133310)3. Yang Wen Huey(133376)4.Joey Lew Hui Lin (133302) Lab 2: Measurement and counting of cells using microscope2.1 Ocular MicrometerIntroductionAn ocular micrometer is a glass disc on which a series of uniformly spaced lines has been inscribed. When ocular micrometer is placed in one of the eyepieces of the microscope， the distance between the etched lines depends upon the objective lens used to view the specimen. In order to determine the precise distance between the lines of an ocular micrometer, a stage micrometer must be used to do the calibration job. The inscribed lines on a stage micrometer are exactly 0.01 mm or 10 μm apart. When the objectives changed, recalibration of the micrometer must be done, . After calibration of the ocular micrometer, the stage micrometer is replaced with a slide containing microorganisms. The dimensions of the cells may then be determined. ObjectiveTo measure and count cells using a microscopeMaterials and ReagentsMicroscope fitted with an ocular micrometerSlide micrometerStained preparation of yeast and bacteria ProcedureThe stage micrometer is placed on the stage.Focus the microscope until the image on the stage micrometer is observed superimposed on the eyepiece scale by using the power 100x objective. Determine how many divisions of the eyepiece scale correspond top a definite number of divisions on the stage scale.The measurement of an eyepiece division in micrometer (μm) is calculated.The process is repeated by using the high-power and oil immersion objective.Each division of the stage micrometer = 10 μm.

• 100 eyepiece divisions = 10 stage divisions = 100 μm,

• 100 eyepiece divisions = 10 stage divisions = 100 μm,

• 100 eyepiece divisions = 10 stage divisions = 100 μm,

• 100 eyepiece divisions = 10 stage divisions = 100 μm,

• 100 eyepiece divisions = 10 stage divisions = 100 μm,

• Then , 1 eyepiece division = 100/100 = 1.0 μm

• Then , 1 eyepiece division = 100/100 = 1.0 μm

• Then , 1 eyepiece division = 100/100 = 1.0 μm

• Then , 1 eyepiece division = 100/100 = 1.0 μm

• Then , 1 eyepiece division = 100/100 = 1.0 μm

     7. The diameter of the field for each objective is calculated and recorded for future reference. 8. The average dimensions (in μm) of a sample of yeast cells is determined. The process is  repeated for a bacterial cell sample. At least 10 observations should be included for your samples.                                                               Image 1Using your 100x objective,e.g (shown here),10 divisions on the ocular micrometer scale equal 0.01mm on the stage micrometer.So each small division on the ocular micrometer=1μm.Distance between ocular micrometer divisions is different for each objective;so each objective must be calibrated with the stage micrometer.                                                               Image 2                                                  Image 3 :  Ocular micrometer                                                 Image 4:Ocular micrometer         Image 5:Stage scale Result

Sample	Ocular Readings(units)	Stage Measurement (μm)
1	2	20
2	2.5	25
3	3	30
4	3	30
5	2.5	25
6	2	20
7	2	20
8	2.5	25
9	3	30
10	2	20

The average =  The total of stage measurement / the total number of specimen           = 245/ 10                              = 24.5µm                                         Image 6 Yeast cells under ocular micrometer2.2 Neubauer Chamber

IntroductionNeubauer chambers are more convenient for counting microbes. The Neubauer is a heavy glass slide with two counting areas separated by a H-shaped trough (see Figure 2.1). A special coverslip is placed over the counting areas and sits a precise distance above them.

Materials and ReagentsSerial dilutions of bacteria culturesNeubauer and coverslip70% ethanolSterile Pasteur pipettes

ProcedureA drop of diluted yeast culture(use 10-3 or 10-4 dilution) is added to the space between the coverslip and the counting chamber by using a sterile Pasteur pipetteCells are allowed to settle for about one minute.The cells are counted in the four corner and centre squares. For a reasonably accurate count, we should have more than 30 cells per area.The neubauer and coverslip are cleaned with ethanol(70%).

CountingThe chamber contains many grids, producing nine major large squares.For calculation purposes, only the middle large square is used.The middle large square has a size of 1 mm x 1 mm and a depth of 0.1 mm.Inside the middle large squares, there are 16 smaller squares, each with the size of 0.25mm x 0.25 mm.The number of yeast cells are calculated in the 16 squares.The number of cells per square is average.Assuming the average number of cells =  Z; 1 mm3 = 0.001 cm3; 1 cm3 = 1 mLCell concentration =  (Z/ 16(0.25mmx0.25mmx0.1mm) cells/mL

ResultZ= 27 Cell Concentration= 27/ 16(0.25mmx0.25mmx0.1mm)      = 270 cells per 0.1mm2

Discussion

1. Ocular micrometer a glass disk that fits in a microscope eyepiece and that has a ruled scale; when calibrated with a slide micrometer, direct measurements of a microscopic object can be made.

1. When using difference magnification, the division of ocular micrometer will has difference value. Thus, it is necessary to calibrate the scale by focusing on a fix and known scale micrometer (a stage micrometer) placed directly on the stage for every magnification to make sure the the accurate of the ocular micrometer.

1. Scan square subdivisions from left to right, up to down and count the cells. Count the cells if they are touching the left or top line of each square ，bottom and right will not be calculated. This can make sure that the cells that touching the lines are not counted twice. Do not count the cell in the bottom row either.

ConclusionOcular micrometer with a known scale stage micrometer , the cell can be measuring easily and accurately especially with oil immersion method.