Microscopic direct counting of microorganisms

, the experimental purpose
to understand the structure of the blood cell counting board, counting principles and counting methods, master the
microscope
direct counting skills.
, experimental principle
to determine the number of
microbial
cells is a lot of methods, usually using a microscopic direct counting method and plate counting method.
microcounting method is suitable for all kinds of pure
culture
suspensions containing single-celled bacteria, if there are germs or impurities, often difficult to distinguish. Larger
yeasts
or mold spores can use blood cell counting boards, while the average bacteria use Petrof Hausser bacterial counting plates. The principles and components of the two counting plates are the same, but the bacterial counting plates are thin and can be observed using an oil mirror. And the blood cell count plate is thick, can not use oil mirror, the bacteria in the lower part of the counting plate is not easy to see.
blood cell counting board is a specially made thick slide with 4 slots and 3 platforms. The platform in the middle is wider, and the middle is divided into two halves by a short horizontal slot, each with a counting area above each half, and there are two scales for the counting area: one is that the count is divided into 16 squares (the large squares are divided into three squares) line separated), and each large square is divided into 25 small squares, the other is a count divided into 25 large squares (the large squares are separated by two lines), and each large square is divided into 16 small squares. But regardless of the structure of the counting area, they all have a common feature, that is, the counting area is composed of 400 small squares.if the edge length of the
counting area is 1mm, the area of the counting area is l mm2, and the area of each small square is 1/400mm2. After the cover glass is covered, the height of the counting area is 0.1mm, so the volume of each counting area is 0.1mm3, and the volume of each small square is 1/4000mm3.
the use of blood cell counting boards, the number of microorganisms in each small square is first measured and then converted to the number of microbial cells per milliliter of bacteria (or per gram of sample).
known: 1mm3 volume: 10 mm×10 mm×10 mm s 1000mm3
so: 1mm3 volume should contain a small number of small squares of 1000mm3/1/4000mm3 x 4×106 small squares, i.e× factor K is 4×106.
In this way: the number of cells per ml of bacteria suspension contains the average number of cells in each small grid (N) × coefficient (K)× bacteria dilution multiply (d)
3, experimental equipment
1. Living material: wine-making yeast (Saccharomyces cerevisiae) slope or culture solution.
2. Equipment: microscope, blood cell counting board, cover slide (22mm×22mm), absorbent paper, counter, dropper, mirror paper.
, the experimental method is
1. Depending on the concentration of bacteria suspension to be measured, the sterile water should be properly diluted (slope is generally diluted to 10-2) to the number of bacteria per small grid.
2. Take a clean blood cell counting board and cover the counting area with a cover glass.
3. Shake the yeast suspension well, draw a little with a dropper, remove a small drop (not too much) from the groove on both sides of the platform in the middle of the counting plate along the lower edge of the cover glass, so that the bacteria suspension fills the counting area with the surface pressure of the liquid, do not make bubbles, and use absorbent paper to suck out the excess bacteria suspended from the trench. Bacteria suspension can also be added directly to the counting area, do not make the two sides of the counting area platform contaminated with bacterial suspension, so as not to cover the slide, resulting in an increase in the depth of the counting area. Then cover the slide (do not cause bubbles).
4. Rest for a moment, clamp the blood cell count board on the loader, find the count area under the low multiplle mirror, and then convert the high multiplile mirror to observe and count. Because the recrout rate of living cells is similar to that of water, the intensity of light should be reduced when observed.