Diluted plate measurement method

First, the experimental purpose
to understand the principle of dilution plate counting, master the application of flat plate
culage
method and mixed plate culture method, to understand bacteria, line bacteria, mold fall characteristics.
II, experimental principle
dilution plate count is based on
microorganisms
on the solid
culture base
formed a single organism, that is, by a single cell to reproduce the culture characteristics of the design of the counting method, that is, a cell represents a single cell. When counting, first of all, the sample to be tested into a uniform series of dilution, as far as possible to disperse the microbial cells in the sample, so that a single cell exists (otherwise a bacterium does not just represent a cell), and then take a certain dilution, a certain amount of dilution inoculated into the plate, so that it is evenly distributed in the plate medium.
cultured, a single cell grows and reproduces to form a bacteria,
statistics
the number of bacteria, you can calculate the number of bacteria in the sample. The number of bacteria calculated by this method is the number of bacteria that grow on the media, so it is also known as the count of live bacteria. Generally used for the inspection of certain finished products (e.g. insecticides, etc.), biological products testing, soil bacteria content determination and food, water pollution levels.
third, experimental equipment
1. Living material: Bacillus thuringiensis.
2. Media: Beef paste protein
agar
media
3. Equipment: 90 ml of sterile water, 9 ml of sterile water, sterile flat dish, lml sterile straw,
balance
, sample bottle, marker pen, glass scraper, etc.
, the experimental method is
1. Preparation of sample dilution
accurately called sample l0g under test, placed in a 250 ml triangular bottle containing 90 ml of sterile water and small glass beads, oscillating 20 min by hand or on a shaker bed, so that microbial cells disperse, quiet Set 20-30s, i.e. into 10-1 diluents, and use 1 ml of sterile straw, absorb 10-1 diluent lml, move into
test tubes containing 9 ml
sterile water, blow Suck 3 times, let the bacteria mix evenly, that is, into 10-2 dilution, and then another sterile straw to absorb 10-2 dilution 1 ml, into a test tube filled with 9 ml of sterile water, also blow three times, that is, into 10 -3 dilution, and so on, continuous dilution, to make 10-4, 10-5, 10-6, 10-7, 10-8, 10-9 and a series of diluted bacteria.
the dilution plate count, the selection of the dilution of the bacteria to be measured should be determined according to the sample. When the number of bacteria to be tested in the sample is high, the dilution should be high and then low. Usually when measuring the number of bacteria containing bacterial agents, 10-7, 10-8, 10-9 dilution is used, when determining the number of soil bacteria, 10-4, 10-5, 10-6 dilution is used to determine the line When the number of bacteria, l0-3, 10-4, 10-5 dilution was used, and 10-2, 10-3, 10-4 dilution was used to determine the number of
fungi
.
2. Tablet inoculation culture
plate inoculation culture has mixed plate culture method and application plate culture method two methods.
(1) mixed plate culture method
the sterile plate on 10-7, 10-8, 10-9 numbers, each number set three repetitions, with sterile straw according to sterile operation requirements to absorb 10-9 diluent each 1 ml into the number 10-Of the 3 plates of 9, the same method absorbs 10-8 diluents each lml into the number 10-8 of the 3 plates, and then absorbs 10-7 diluents each lml into the number 10-7 of the 3 plates (from low concentration to high concentration, straw can not be replaced). Then pour in the 9 plates have melted and cooled to 45-50 degrees C of bacterial medium, gently turn the plate, so that the liquid and medium mixed evenly, cold doubt after upside down, temperature-appropriate culture. Count by the time the bacteria grow behind.
(2) Applying the plate counting method
Applying the plate counting method is basically the same as the mixing method, the difference is that the medium is melted and poured into the sterile plate while it is hot, to be solidified after the number, and then with a sterile straw to absorb 0.1 ml of bacteria liquid pair inoculated on different dilution numbers on the agar plate (three repetitions each number). Then use a sterile spatula to apply the bacteria liquid evenly on the plate, each dilution with a sterilization spatula, replace the dilution need to burn the spatula to sterilise. When applying from low to high concentrations, the scraper can also be replaced. Place the coated plate flat on the table for 20-30min, allow the liquid to penetrate into the culture, then turn the plate upside down, keep warm and culture, until the bacteria grow behind can be counted.