Common detection methods of microbial growth

1. 1 volume measurement method: also known as mycelium concentration measurement method.

By measuring the number of hyphae in a certain volume of culture medium, the growth status of microorganisms can be reflected. The method is to take a certain amount of culture solution to be tested (such as 10 ml) and put it in a graduated centrifuge tube, set a certain centrifugation time (such as 5min) and rotation speed (such as 5000rpm), then pour out the supernatant after centrifugation, and measure the volume of the supernatant as V and the mycelium concentration as (10-V)/65438. The determination of mycelium concentration is an important monitoring index for microbial growth in large-scale industrial fermentation production. This method is extensive, simple and rapid, but it needs to set consistent treatment conditions, otherwise the deviation will be great, because some solid nutrients are mixed in centrifugal precipitation, and the results will be somewhat biased.

1.2 dry weight method:

It can be determined by centrifugation or filtration. Generally, the dry weight is 20% of 10- wet weight. Centrifugation method is to pour a certain volume of culture solution to be detected into a centrifuge tube, set a certain centrifugation time and rotation speed, centrifuge, centrifuge with clean water for 1-5 times, and dry. Drying can be carried out in an oven at 105℃ or 100℃, or by infrared rays, or by vacuum drying at 80℃ or 40℃, and then weighing. If filtration method is used, filamentous fungi can be filtered by filter paper, and bacteria can be filtered by filter membrane such as cellulose acetate membrane. After filtration, it was washed with a little water and dried in vacuum at 40℃. It's called dry propagation. It's complicated. Usually, when the obtained microbial products are thallus, this method is often used, such as active dry yeast (ADY), some feeds and fertilizers with microbial thallus as active substances.

1.3 turbidimetry:

The growth of microorganisms leads to the increase of culture turbidity. Using ultraviolet spectrophotometer to measure the light absorption value of a certain wavelength to judge the growth of microorganisms. A special triangular flask with a side arm can be used to regularly track the growth of bacteria in the culture. Insert the side arm into the hole of the colorimetric seat of the photoelectric colorimeter, and the growth of the side arm can be determined at any time without taking the bacterial solution. This method is mainly used to monitor the growth of bacteria in fermentation industry. For example, I used the UV-Vis spectrophotometer of UNICO company, and measured the absorbance value OD600 of fermentation broth regularly with a colorimetric tube at 600nm wavelength to monitor the growth and induction time of Escherichia coli.

1.4 mycelium length measurement method:

For filamentous fungi and some actinomycetes, the length of mycelium growth in a certain period of time can be measured on the culture medium, or it can be obtained by using a thin glass tube with one end open and graduated.

Culture medium, standing, inoculating microorganisms at one end of the opening, and recording the growth length of mycelium after a period of time to measure the growth of filamentous microorganisms.

Second, microbial counting method

2. 1 blood cell count plate method:

Blood cell counting plate is a thick glass plate with special structural dimensions and thickness. There are four grooves and two ridges on the slide, and there is a short horizontal groove and two platforms in the center. The surface of the two ridges is 0. 1mm higher than that of the two platforms. Each platform is engraved with grids of different specifications, and 400 small squares are engraved on the center area of 0. 1mm2. By observing with an oil mirror and counting the number of microorganisms in a large cell, the number of bacteria contained in 1 ml bacterial liquid can be calculated. This method is simple, intuitive and fast, but it is only suitable for counting spores produced by single-celled microorganisms or filamentous microorganisms, and the result is the total number of bacteria including dead cells.

2.2 Dyeing counting method:

In order to make up for the deficiency that some microorganisms are not easy to observe and count under the oil mirror, and the blood cell counting plate method can not directly distinguish dead cells from living cells, people invented the dyeing counting method. With the help of different dyes, it is more convenient to count live bacteria under the microscope. For example, methylene blue staining solution can be used to count the number of yeast living cells. After staining, living cells are colorless and dead cells are blue.

2.3 Proportional counting method:

The "cell concentration" of the unknown bacterial solution can be obtained by evenly mixing the liquid with known particle concentration (such as mold spores or red blood cells) with the bacterial solution with the cell concentration to be measured in a certain proportion and counting their respective numbers in the field of view of the microscope. This counting method is quite extensive. And it is necessary to prepare a suspension with a known particle concentration as a standard.

2.4 liquid dilution method:

Unknown bacterial samples were continuously diluted 10 times. According to the estimation, 5 ml samples were taken from three consecutive dilutions of the most suitable 10 times, and then inoculated into three groups of test tubes containing *** 15 medium. After culture, record the number of test tubes grown at each dilution, and then check the maximum probability table MPN(mostparylnumber). This method is often used to detect microorganisms in food, such as microbial limit inspection of drinking water and milk.

2.5 Plate colony counting method:

This is one of the most commonly used methods for counting live bacteria. Carrying out gradient dilution on the bacterial liquid to be detected, taking a certain volume of diluted bacterial liquid and uniformly mixing the diluted bacterial liquid with an appropriate solid culture medium before curing, or coating the bacterial liquid on a solidified solid culture medium plate. After incubation, the bacterial count of the original bacterial solution can be calculated by multiplying the number of colonies appearing on the plate by the dilution of the bacterial solution. Generally, 50-500 colonies appear on the plate with a diameter of 9cm. But the method is troublesome, and the operator needs skilled technology. The plate colony counting method can not only obtain the number of viable bacteria in the bacterial solution, but also separate the bacteria in the cultured bacterial solution at one time to obtain monoclonal antibodies.

2.6 Test paper method:

Based on the plate counting method, a small commercial product for fast counting is developed. There are small and thick filter paper sheets, agar sheets and so on. Absorb appropriate culture medium on filter paper and agar, add active indicator 2,3,5-triphenyltetrazole chloride (TTC, colorless) to soak the test bacteria solution, and then culture it in sealed packaging bags. After short-term culture, rose-colored microcolonies with a certain density appear on the filter paper, and the bacterial content of the sample can be estimated by comparing the spectrum on the standard paper color plate. The test paper counting method is fast and accurate, which avoids the human operation error of the plate counting method.

2.7 membrane filtration method:

A certain volume of bacteria-containing samples were filtered with a special filter membrane, stained with orange, and the fluorescence of cells was observed under an ultraviolet microscope. Living cells emit orange fluorescence, while dead cells emit green fluorescence.

2.8 Physiological index method:

The growth of microorganisms is accompanied by a series of changes in physiological indexes, such as pH, nitrogen content, sugar content and gas production of fermentation broth. There are many physiological indexes parallel to the growth amount, which can be used as relative values for growth measurement.

2.9 Determination of nitrogen content:

The nitrogen content of most bacteria is 12.5% dry weight, yeast is 7.5%, and mold is 6.0%. According to the nitrogen content ×6.25, the crude protein content can be determined. There are many methods to determine nitrogen content, such as sulfuric acid digestion, perchloric acid digestion, iodic acid digestion, phosphoric acid digestion and Du Masi method. Du Masi's method for measuring N2 gas is to mix the sample with CuO, heat it in CO2 gas flow to generate nitrogen, collect it in a respirometer, and measure the amount of N2 after CO2 is absorbed by KOH.

2. Determination of10 carbon content:

A small amount of biomaterial (0.2-2.0 mg dry weight) is mixed into 1 ml water or inorganic buffer, heated with 2 ml 2% K2Cr2O3 solution at 1000℃ for 30 minutes, and then cooled. Add water to dilute to 5 ml, read the absorbance value at 580nm wavelength, and calculate the growth amount. It is necessary to use reagents as blank control and standard samples as standard curves.

2. 1 1 determination method of reducing sugar:

Reducing sugar usually refers to monosaccharide or oligosaccharide, which can be directly utilized by microorganisms. The determination of reducing sugar can indirectly reflect the growth of microorganisms, and is often used for routine monitoring of microbial growth in large-scale industrial fermentation production. The method is: centrifuge the fermentation broth, collect the supernatant, add Fehling reagent, boil in boiling water bath for 3 minutes, take it out and add a little hydrochloric acid for acidification, add starch solution near the end point, continue to add Na2S2O3 to the end point, and look up the table to read the reducing sugar content.

2. Determination of12 amino nitrogen:

The method is to centrifuge the fermentation broth, collect the supernatant, add methyl red and hydrochloric acid as indicators, add 0.02N NaOH to adjust the color until it just fades, add 18% neutral formaldehyde as substrate, react for a few minutes, add 0.02N to change color, and calculate the content of amino nitrogen according to the amount of NaOH. According to the content of amino nitrogen in the culture medium, the growth status of microorganisms can be indirectly reflected.

2. 13 determination of other physiological substances:

The contents of P, DNA, RNA, ATP, NAM and other indicators, such as acid production, gas production, CO2 production (using labeled glucose as substrate), oxygen consumption, viscosity and heat production, can be used to determine growth. The growth of microorganisms can also be reflected according to the change of substrate concentration, final gas production and microbial activity before and after the reaction. For example, in the fermentation production of BMP-2, I always judge the growth of bacteria by monitoring the changes of dissolved oxygen and pH value.

Extension: Modern Definition of Microorganism

It is difficult to see clearly with the naked eye, and it needs to be observed with the help of an optical microscope or an electron microscope. Microorganisms include bacteria, viruses, fungi and some algae. (But some microorganisms are visible to the naked eye, such as mushrooms and ganoderma lucidum, which belong to fungi. ) Virus is a kind of "acellular organism" composed of nucleic acid and protein, but its survival depends on living cells. According to the different environment, it can be divided into space microorganisms and marine microorganisms. According to the cell structure, it can be divided into prokaryotic microorganisms and eukaryotic microorganisms.

Main characteristics of microorganisms

Small body and big face

The smaller the volume of an object is cut, the larger the relative surface area is. Microorganisms are very small, such as a typical cocci, with a volume of about 1mm, but a large surface area. This characteristic is also the basis of giving microorganisms other characteristics such as fast metabolism.

Smoke more and turn faster.

Microorganisms usually have extremely efficient biochemical transformation ability. According to the research, lactic acid bacteria can decompose lactose which is 1 000-10000 times its own weight in1hour, and the protein synthesis ability of Candida utilis is100 times that of soybean protein.

Rapid growth and reproduction

Compared with large animals, microorganisms have a very high growth and reproduction speed. Escherichia coli can reproduce 12.5-20 minutes/time. Let's calculate, 1 Escherichia coli divided by 1 times in 20 minutes, 1 hour divided by 3 times, 1 day and night divided by 24 times = 72 times, which can produce about 472,236,500 trillion (72 power of 2), which is a very huge number. But in fact, due to various conditions, such as lack of nutrition, intensified competition, deterioration of living environment and other reasons, microorganisms can not fully achieve this exponential growth. It is known that the optimum pH range of most microorganisms is around 7.0 (6.6~7.5), and some of them are below 4.0.

This characteristic of microorganism makes it widely used in industry, such as fermentation, single cell protein and so on. Microorganisms are indispensable good friends of human beings.

Strong adaptability and variability

Widely distributed and diverse.

The influence of microorganisms on our lives

One of the most important effects of microorganisms on human beings is the prevalence of infectious diseases. 50% of human diseases are caused by viruses. The history of human diseases caused by microorganisms is also the history of human struggle against it. Great progress has been made in the prevention and treatment of diseases, but new and reappearing microbial infections continue to occur, for example, a large number of viral diseases have been lacking effective therapeutic drugs. The pathogenesis of some diseases is still unclear. The abuse of a large number of broad-spectrum antibiotics has caused strong selection pressure, which has caused many strains to mutate and produce drug resistance, posing a new threat to human health. Some segmental viruses can mutate through recombination or rearrangement, and the most typical example is influenza virus. Every time an outbreak of influenza occurs, the influenza virus will mutate from the strain that caused the infection last time. This rapid mutation has caused great obstacles to the design and treatment of vaccines. The emergence of drug-resistant mycobacterium tuberculosis has made the tuberculosis infection that was almost controlled rampant in the world.

There are many kinds of microorganisms, some of which are corrupt, that is, they cause bad changes in food odor and tissue structure. Of course, some microorganisms are beneficial. They can be used to produce cheese, bread, pickles, beer and wine. Microorganisms are so small that they must be magnified with a microscope to see them. For example, for medium-sized bacteria, 1000 is only as big as a period.

Microorganisms can cause diseases, which will cause food, cloth, leather and other moldy rot, but microorganisms also have a beneficial side. It was Fleming who first discovered penicillin from Penicillium which inhibited the growth of other bacteria, which was an epoch-making discovery in the medical field. Later, a large number of antibiotics were screened from the metabolites of actinomycetes. The use of antibiotics saved countless lives in World War II. Some microorganisms are widely used in industrial fermentation to produce ethanol, food and various enzyme preparations. Some microorganisms can degrade plastics and treat wastewater and waste gas. , and has great potential of renewable resources, known as environmental microorganisms; Some microorganisms can survive in extreme environments such as high temperature, low temperature, high salt, high alkali and high radiation, and some microorganisms still exist. It seems that many microorganisms have been discovered, but in fact, due to the limitation of technical means such as culture methods, the microorganisms discovered by human beings today only account for a small part of the existing microorganisms in nature.

The interaction mechanism between microorganisms is also quite mysterious. For example, there are a large number of bacteria in the intestines of healthy people, which are called normal flora, including hundreds of bacteria. In the intestinal environment, these bacteria are interdependent and mutually beneficial. The decomposition and absorption of food, toxic substances and even drugs, the role of flora in these processes and the interaction mechanism between bacteria are still unknown. Once the flora is out of balance, it will cause diarrhea.

With the medical research entering the molecular level, people are more and more familiar with the technical terms such as genes and genetic materials. It is recognized that genetic information determines the life characteristics of organisms, including external morphology and life activities, and the genome of organisms is the carrier of these genetic information. Therefore, understanding the genetic information carried by the genome of an organism will be of great help to reveal the origin and mystery of life.

Industrial microorganisms involve food, pharmacy, metallurgy, mining, petroleum, leather, light chemical industry and many other industries. Production of antibiotics, butanol, vitamin C and preparation of some flavor foods by microbial fermentation; Some special microbial enzymes are involved in leather depilation, metallurgy, oil extraction and mining, and even directly used as additives for washing powder. In addition, some microbial metabolites can be widely used in agricultural production as natural microbial pesticides. By studying the genome of Bacillus subtilis, a series of genes related to the production of antibiotics and important industrial enzymes were found. As an important microecological regulator, lactic acid bacteria participate in the food fermentation process. Genomic research on lactic acid bacteria will help to find key functional genes, and then transform the strain to make it more suitable for industrial production. The genome research of Gluconobacter oxydans, the key strain in the two-step fermentation process of vitamin C in China, will find important metabolic functional genes related to vitamin C production on the premise of genome sequencing, and realize the construction of new engineering strains through genetic engineering transformation, simplify production steps, reduce production costs, and then greatly improve economic benefits. The genome research of industrial microorganisms has continuously found new special enzyme genes and functional genes related to important metabolic processes and metabolites, and applied them to production and the transformation of traditional industries and processes, which has promoted the rapid development of modern biotechnology.

The pathogen of commercial crop citrus is the first plant pathogenic microorganism in the world to publish its full sequence. There are also some agricultural microorganisms that are very important in taxonomy, physiology and economic value, such as Erwinia carotovora, Pseudomonas spp. and Xanthomonas spp., which are being studied in China. Recently, the complete sequence of nitrogen-fixing rhizobia in plants has just been determined. Drawing on the mature scheme of screening therapeutic drugs from the genome information of human pathogenic microorganisms, it can be tentatively applied to plant pathogens. Especially citrus pathogens need insect vectors to complete their life cycle. Only by finding virulence-related factors and resistance targets through genetic research can more effective control strategies be formulated. The analysis of all genetic information of nitrogen-fixing bacteria is also of great significance for developing and utilizing its key nitrogen-fixing genes and improving crop yield and quality. [ 10]

Microorganisms that can grow in extreme environments are called extremophiles, also known as extremophiles. Extreme microorganisms have strong adaptability to extreme environments. The study of extreme microbial genome is helpful to study the adaptability of microorganisms under extreme conditions at the molecular level and deepen the understanding of the nature of life.