Introduction to the determination of microbial biomass and growth curve

Experimental principle

We know that microorganisms are characterized by rapid growth and rapid reproduction. The increase of individual cells of single-cell microorganisms such as bacteria and yeast is limited. The growth of cells is limited to a certain extent. The number has increased. When the bacteria grow vigorously, they can breed for a few dozen minutes.
Therefore, their growth is often manifested through reproduction, which is essentially a growth sign of the number of population cells. The growth of filamentous microorganisms such as actinomycetes and molds is mainly characterized by the elongation and branching of hyphae. They are intertwined and it is difficult to distinguish the boundaries between individuals and individuals, so it usually grows with the weight of hyphae (cell quality). Increase) or mycelial growth rate indirectly to measure growth. Current methods for determining the number of microorganisms are direct and indirect. The direct method includes a blood cell counting plate method, a smear staining method, a turbidimetric method, and the like; the indirect method includes a plate colony counting method, a liquid dilution method, a membrane filtration counting method, and the like. The method for measuring the mass of the cells includes a nitrogen determination method, a DNA method, a measurement of a cell dry weight method, a measurement of a wet cell weight method, and a physiological index measurement method.
In order to learn the growth law of microorganisms and improve the characteristics of microbial growth and rapid reproduction, yeast, actinomycetes and cucumber Fusarium oxysporum were used as experimental materials. The experiment used dry/wet weight method, blood cell counting method for direct counting and turbidity. The method and the method of measuring the length of mycelium, the growth of actinomycetes and cucumber Fusarium oxysporum and the growth curve of Fusarium oxysporum f. sp., and the yeast growth standard curve (cell density - OD420) were drawn.

Experimental reagent
Medium: Beef extract peptone liquid medium (broth culture medium) High's No.1 medium (liquid/solid) Potato sucrose medium (PDA liquid/solid)
Reagents: sterile water

Laboratory equipment
Oscillator Â
Blood cell counting board (0.10mm 1/400mm2 16Ã—25)
Electric furnace constant temperature culture shaker ordinary centrifuge ultra-clean workbench Â
Constant temperature incubator Â
Portable high pressure steam sterilizer Â
Optical microscope Â
721 spectrophotometer Â
Cuvette Â
Brinell funnel Â
Oven Â
Puncher Â
Vaccination ring Â
Qualitative filter paper Â
Ruler, etc.

Experimental Materials
Strain: Actinomycetes Saccharomyces Cerevisiae Fusarium oxysporum

Experimental procedure
1. Actinomycete biomass determination
(1) Determination of biomass of hyphae - dry/wet weight method
The actinomycetes isolated from the soil were densely streaked on a Gaussian plate 1 and cultured for one week. Use a puncher to lay down several bacteria blocks, take 3 pieces of bacteria into 3 bottles of 50ml/250ml Gao's No.1 liquid medium, put 1 piece of bacteria into 50ml/250ml sterile water, shake bottle at 30Â°C 200rpm After one week of culture, it was filtered with a qualitative filter paper, and the hyphae were collected. After the wet weight was measured, the hyphae were placed in an oven at 80 Â° C and dried to constant weight, and the weight of the hyphae measured was the dry weight.
2. Determination of the growth of Fusarium oxysporum
(1) Mycelium length measurement method
The growth rate of Fusarium oxysporum f. sp. was estimated by measuring the diameter of colony growth.
Pick a small medium with hyphae of Fusarium oxysporum f. sp. inoculated in the center of the PDA plate. After one week of culture, inoculate with a 0.8 cm diameter sterile puncher to the center of the 9.0 cm diameter PDA plate, every other week in the next week. The growth length of the hyphae was measured at 12 hours until the hyphae covered the whole dish. The average growth rate of the hyphae was determined, and a growth curve was drawn therefrom.
(2) Determination of biomass of hyphae - dry/wet weight method
The PDA plate of Cucumber Fusarium oxysporum cultured for one week was cut into several pieces with a puncher, and three pieces of the bacteria were placed in three bottles of 50 ml/250 ml PDA liquid medium, and one piece of the bacteria was placed in 50 ml/250 ml of sterile water. After 30 Â°C 200 rpm shake flask culture for one week, filter with qualitative filter paper, collect hyphae, measure the wet weight, put the hyphae in an oven at 80 Â° C to dry to constant weight, and then measure the weight of the hyphae as dry weight .
3. Determination of yeast growth
(1) Determination of OD value of yeast cell suspension - turbidimetry
The yeast was inoculated into a broth culture solution (50 ml/250 ml), one vial of each bottle, a total of 2 bottles, and shaken at 28 Â° C for 125 rpm in a shake flask for two weeks. The culture solution was dispensed into a 10 ml centrifuge tube and centrifuged at 5000 r/min for 5-6 minutes. The supernatant was discarded, resuspended in sterile water, centrifuged, and the supernatant was discarded, and repeated twice to remove the remaining solution.
The precipitates were combined and diluted to a dilution of 6 dilutions with sterile water to measure the OD value at 420 nm (the effective measurement range of the 721 spectrophotometer was 0.1-0.65).
(2) Determination of cell concentration of yeast suspension - blood cell count plate count
After washing the blood cell counting plate, cover the cover glass on the counting chamber, and use a fine-mouthed pipette to blow up one of the diluted bacterial liquids back and forth several times, so that the bacterial liquid is fully mixed and immediately absorb a small amount of bacterial liquid and add it to the cover. On the edge of the slide, allow the bacterial liquid to infiltrate into the counting chamber from the gap between the cover glass and the counting plate (no air bubbles in the counting chamber). The coverslip is lightly pressed to prevent the cover glass from being lifted up due to excessive bacterial liquid and changing the volume of the counting chamber.
After standing for a while, after the natural sedimentation of the cells is stable, first use the low power microscope to find the position of the counting chamber (the field of view should be dimmed), then find it and move it to the center of the field of view, then change it to observe and count. After counting, wash the counter. Each dilution is counted once.
Counting principle: In order to reduce the error, it is common to take 4 squares in 4 corners and 1 square in the center to count, and finally take the average. In addition: the count does not count, the left is not counted right, the count must have a certain path.
A standard curve is drawn based on the OD value and the yeast cell blood cell count value.

Precautions
The growth and reproduction of microorganisms is a comprehensive reflection of the physiological and metabolic states under the interaction of various environmental factors inside and outside. Therefore, the data on growth and reproduction can be used as an important indicator for studying various physiological, biochemical and genetic problems. Meanwhile, microorganisms Various applications in production practice or human control of harmful microorganisms such as pathogenicity and mildew are also closely related to their growth and reproduction.
This experimental method has its significant advantages and has some shortcomings:
1. The blood cell counting plate is a commonly used direct micro-technical method, which has the advantages of being intuitive, quick and easy to operate, but it is laborious, hurts the eyes, and requires high concentration of the suspension, generally not too low or too high (usually greater than 10) ~6 / m), the OD value should be between 0.1-0.65, which is difficult to grasp. Moreover, this method can not measure the number of viable bacteria. If a staining solution such as phenol blue is used, the dead cells can be dyed blue, and the living cells are not stained, thereby measuring the number of viable cells.
2. The wet weight method and the dry weight method can reflect the amount of biomass in the fermentation broth, but it does not reflect the physiological state of the hyphae, the degree of aging, or even the differentiation of cells.
3. It is simple and convenient to measure the growth status of the filamentous fungi grown by radiation, but it seems to be not very applicable.
Therefore, each method has its own advantages and limitations. Only after considering the relationship between these factors and the problems that need to be solved, can we choose the better ones or several methods at the same time.
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