Directo conteo de microorganismos con un microscopio
Experimental principio
Direct counting under a microscope using a hemocytometer is a commonly used microbial counting method. The advantage of this method is that it is intuitive and fast. Place the appropriately diluted bacterial suspension (or spore suspension) in the counting chamber between the slide and the cover glass of the hemocytometer, and count under the microscope. Since the volume of the counting chamber is fixed (0. 1mm2), it can be converted into the total number of microorganismos per unit volume according to the number of microorganismos observados under the microscope. Because this method counts the sum of live and dead bacteria, it is also called the total bacteria count method.
A hemocitómetro es usualmente a especial vidrio diapositiva on que cuatro ranuras forman tres plataformas. La plataforma en el medio es dividido en dos mitades por a corto horizontal ranura. A cuadrícula es grabado en la plataforma on each side. Each grid is divided into nine large squares. The large square in the middle is the counting room. Microorganisms are counted in the counting chamber. The structure of the blood cell counting board is shown in Figura Ⅷ-1.
The scale of the counting room generally has two specifications, one is that a large square is divided into 16 middle squares, and each middle square is divided into 25 small squares (Figure Ⅷ-2); the other is a large square. The square is divided into 25 middle squares, and each middle square is divided into 16 small squares (Figure Ⅷ{{5}% 7d, C). Pero no importa qué kind of counting board it is, the number of small squares in each large square is the same, that is, 16×25=400 7d small squares, as shown in Figure Ⅷ-2.
The side length of each large square is 1 mm, and the area of each large square is 1 mm2. After the cover glass is covered, the height between the slide glass and the cover glass is 0.1 mm, so the volume of the counting chamber is 0.1mm3.
When counting, usually count the total number of bacteria in the five middle grids, then obtain the average value of each middle grid, and then multiply by 16 or 25 to get the total number of bacteria in a large grid, and then Converted to the total number of bacteria in 1ml bacterial solution.
Laboratorio equipo
Hemocitómetro, microscopio, coverslip, estéril capilar;
Experimental Materiales
Saccharomyces cerevisiae suspensión
1. dilución
Propiamente diluir el Saccharomyces cerevisiae suspensión. Si el bacteriano solución es no esgrueso, it hace no necesita ser diluido.
2. Microscopic counting room
Antes sumando muestras, realizar a microscópica inspección de la cámara conteo de la conteo placa. Si hay suciedad, it necesita a ser limpiado antes conteo.
3. add sample
Cover the clean and dry hemocytometer plate with a cover glass, and then use a sterile fine-mouth dropper to drop a small drop of the diluted Saccharomyces cerevisiae solution from the edge of the cover glass (not too much), so that the bacteria solution is close to the gap along the gap. Capillary osmosis enters the counting chamber by itself, and the general counting chamber can be filled with bacterial liquid. Be careful not to have air bubbles.
4. microscopio recuento
Después de descansar durante 5 minutos , lugar el hemocitómetro en la etapa de el microscopio, primero uso a bajo potencia lente para encontrar la ubicación de la cámara conteo, y luego cambiar a alto potencia lente para contar. Si es es encontrado que la solución bacteriana es demasiado gruesa o demasiado delgada antes de contar , es necesaria volver a ajustar la dilución antes de contar. Generalmente, la muestra dilución requiere acerca de {% 7b2}} bacteria in each cell. Each counting chamber selects the bacteria in 5 middle grids (optional 4 corners and central grids) for counting. The bacteria on the grid line are generally only counted on the upper and right lines. In case of yeast budding, when the size of the bud body reaches half of the mother cell, count two bacteria. Count a sample to calculate the bacterial content of the sample desde los valores contados en los dos contando cámaras.
5. Cleaning the Hemocytometer
