Preparation of a certain concentration of bacterial suspension

gardener Gloria 1983, lphare recommends the Macbeth turbidity method.

Microbiology
experimental guidance provided by
Microbiology 2006 (written by Huang Wenfang, Zhang Song, School of Life Sciences, South China Normal University) is described in detail in the determination of the number of microorganisms in the experiment.
the number of microorganisms
the growth of bacterial populations is reflected in an increase in the number of cells or an increase in the number of cell matter. The methods for determining the number of cells include
microscope
direct counting, plate counting, photobidiment by spectrophotometer, max or number of most probable MPN, and membrane
filtration
(membrane filter).
methods used to determine cell matter are cell dry weight measurements, the content of certain components of cells such as nitrogen, RNA and
DNA
of dna, metabolites, etc. In short, there are many methods to determine the growth of microorganisms, each with advantages and disadvantages, the work should be selected according to the specific requirements.
this experiment mainly introduces the direct microscope counting method, flat-panel bacterioscopic counting method and photoelectrelectroric turbidity counting method, which are commonly used in production and scientific research.
a microscope direct counting method
(i) the purpose of
1. Clarify the
of blood
cell counting and plate counting.
2． Learn how to count microorganisms using blood cell counting plates.
(ii) Basic Principles
The microscope direct counting method is a simple, fast and intuitive way to place the suspension of a small sample to be measured on a special slide with a defined area and volume (also known as a fungicide) and count directly under the microscope. At present, commonly used bacteria at home and abroad are: blood cell counting board, Peteroff-Hauser bacteria meter and Hawksley bacteria, they can be used for
yeast
, bacteria, mold spores and other suspension counting, the basic principle is the same.
the 12th microscope, with a total volume of 0.02mm3 and a distance of only 0.02mm between the cover glass and the carrier, can observe and count smaller cells such as bacteria with an oil-soaked object lens. In addition to using these microscopes, there is an estimation method for directly observing the ratio of smear area to field of view under a microscope, which is generally used for bacteriological examination of cow's milk. The advantage of microscope direct counting is that it is intuitive, fast and simple to operate. However, the disadvantage of this method is that the measured results are usually the sum of dead and living bacteria. There are already some ways to overcome this shortcoming, such as combining live bacteria chromosome
culture
(short time) and addition of cell division inhibitors to achieve the goal of counting only live bacteria. In this experiment, the blood cell counting board was used as an example for direct microscope counting. The other two ways of using fungi can be seen in the instructions of each manufacturer.
a commonly used method of microbial counting by using blood cell counting plates to count directly under a microscope. The counting board is a special slide, which consists of four slots on the three platforms, the middle wide platform is divided into two halves by a short horizontal slot, each side of the platform has a grid, each grid is divided into nine squares, the middle of the large square is the counting room. The scale of the counting room generally has two specifications, one is a large square divided into 25 Squares, and each Chinese grid is divided into 16 small squares; Each large square edge is lmm long, then each large square area is lm2, covered with a slide, the height between the cover slide and the slide is 0.lmm, so the volume of the counting chamber is 0.lmm3 (one in 10,000) Ml)
count, usually count the total number of bacteria in five Chinese cells, and then find the average of each Chinese grid, and then multiply by 25 or 16, to arrive at the total number of bacteria in a large square, and then converted into the total number of bacteria in lmL bacteria.
the total number of bacteria in the five Chinese cells is A, the dilution multiplile of the bacteria is B, if it is the counting plate of 25 Chinese cells, then the total number of bacteria in the
1mL bacteria solution is A/5×25×104×B is 50,000A.B (each) )
same reason, if it is a counting board of 16 Chinese squares, then the total number of bacteria in the
1mL bacteria solution is A/5×16×104×B is 32000A-B (one)
(iii) equipment
1. Bacteria Brewing Yeast
2. Instruments or other utensils Blood cell counting plates, microscopes, cover slides, sterile capillary droppers.
(iv) operation steps
l. Bacterial suspension preparation
sterile physiological saline to make wine yeast into a proper concentration of bacteria suspension
2. The mirror check
the counting room of the counting board before the sample is added. If there is dirt, it needs to be cleaned and blow-dried before counting.
3． Add sample
will clean
driding
blood cell counting plate cover slide, and then use sterile capillary droppers will shake the wine yeast suspension from the edge of the cover glass drop a small drop, so that the bacteria along the gap by capillary penetration automatically into the counting room, the general counting room can be filled with fungus.
to shake the bacteria first when sampling, and the counting room should not have bubbles when sampling.
4． The microscope count
is 5min stationary after the addition, and then the blood cell counting plate is placed on the microscope stage, first using a low multiplile mirror to find the location of the counting chamber, and then replaced with a high multiplile mirror to count.
Adjust the strength of the microscope light appropriate, for the microscope with a reflector light also pay attention to the light do not side, otherwise the field of view is not easy to clearly count the chamber square line, or see only vertical or only horizontal lines.
if the fluid is found to be too thick or too thin before counting, the dilution needs to be re-adjusted before counting. General sample dilution requirements of about 5 to 10 bacteria per small gig is appropriate. Each counting chamber selects 5 middle cells (optional 4 corners and one middle grid in the center) to count the bacteria. Bacteria located on the grid are generally only above and on the right line. In the event of yeast germination, when the size of the bud reaches half that of the mother cell, it is counted as two bacterial bodies. Counting a sample is calculated from the average values calculated in the two counting chambers to calculate the bacteria content of the sample.
5． Wash the blood cell counting plate
After use, rinse the blood cell counting board with water from the tap, do not wash it with hard objects, dry itself after washing it, or dry it with a hair dryer. A mirror examination to see if there are any residual bacteria or other sediments in each small gig. If not clean, it must be washed repeatedly until it is clean.