Sterile Diluents Commonly Used in Experiment on Microbiology

Most cells are suitable for growth in the pH range of 7.2-7.4, but different kinds of cells have different requirements for pH value, and the optimal pH value of the same cell at different growth stages is also different. The primary cultured cells have strict requirements on pH value, while the subculture cells have looser requirements on pH value. Generally speaking, the tolerance of cells to acidic environment is stronger than that of alkaline environment.During the culture process, strictly controlling the pH value of the nutrient solution is conducive to the growth of cells. The more vigorously the cell grows, the more active the metabolism is, and the faster the pH value changes. The retention of metabolites makes the culture fluid turn sour and yellow, which is harmful to cell growth. Therefore, a certain amount of buffer solution is usually added into the culture solution to keep the pH value relatively stable in a certain range.

Balanced Salt Solution (BSS) is consistent with the pH value, osmotic pressure and other environmental conditions under the cell growth state. It has the functions of maintaining osmotic pressure, controlling acid-base balance, supplying energy and inorganic salt components necessary for cell survival and metabolism, which can meet the basic needs of cell survival and maintenance of certain metabolism in vitro experiments. Cells can survive in BSS for several hours, and the formulation of BSS is simple and the cost is low. Therefore, BSS has become the basic solution for the preparation of culture solutions for various tissue cells, and is also a common solution for tissue cells in experimental operations such as vitro temporary preservation, separation, purification and washing.

Phosphate buffer solution(PBS) is one of the most commonly used balanced salt solutions (BSS). It is composed of a mixture solution of monohydrogen phosphate and dihydrogen phosphate, in which monohydrogen phosphate is alkaline and dihydrogen phosphate is acidic. When microorganisms secrete acidic substances, they will react with monohydrogen phosphate to form dihydrogen phosphate; when they secrete alkaline substances, they will react with dihydrogen phosphate to form monohydrogen phosphate. In this way, the whole system can be maintained in a stable pH range. Because phosphate buffer solution is nearly neutral, it has the strongest buffering power in the physiological pH range with relatively low cost. It has become the most widely used inorganic salt buffer solution in the field of life science, and is also one of the popularly used buffers necessary for amynology, cytobiology and molecular biology laboratories.

0.85% physiological salt solution: It can keep the consistent osmotic pressure inside and outside the cell, and help to avoid the damage of cell wall, which will affect the growth and reproduction of bacteria; prevent cell rupture and keep cells in normal physiological balance in a liquid environment; avoid the death of microorganisms due to water loss or excessive water absorption during operation. Although its requirement of total bacterial count is not too high, physiological salt solution plays a certain role in maintaining osmotic pressure, especially in the detection of dying bacteria. However, physiological salt solution is not suitable under any circumstances. It is not suitable to use physiological salt solution but distilled water while testing samples with high salt content (such as roast eel, soy sauce). The PBS phosphate buffer is recommended by SN (inspection standard for export food industry) to detect the total bacterial count, but it is not explicitly proposed in GB, it generally recommends physiological salt solution. Because the preparation of phosphate buffer diluent is complex, physiological salt solution is normally used to detect the general samples, and distilled water is used when the sample is with high salt content.

Sterile water is usually distilled water after sterilization. Distilled water is used as the sterile diluent in mould and yeast tests. GB 4789.15-1994 uses over-salt Chase medium, dilutes the sample and then uses normal saline, which is easy to mess up the salt concentration.Therefore, the standard setter chose distilled water. In fact, distilled water is harmful to both single-celled prokaryotes and eukaryote. Since GB/T 4789.15-2003, this medium has been deleted, but the distilled water has not been changed. In the specific operation, it is better not to use distilled water to dilute the sample, because a sample often needs to be tested for bacteria and fungi at the same time, and it is impossible to be weighed and then diluted separately.

Peptone is a complex mixture of peptone, peptide and amino acids after enzyme hydrolysis of animal and vegetable protein. It is an ampholyte that is highly hygroscopic, soluble in water and has a buffering effect. Some scholars substitute peptone water for phosphate or physiological salt solution in their own experiments, in order to keep the bacteria in the sample active during the dilution process and avoid death, but the dilution time must be controlled within 15 minutes.