Culture of Microorganisms: 6 Laboratory Instruments

The following points highlight the six common laboratory instruments used in the culture of microorganisms. The instruments are: 1. Class-Wares 2. Colony Counter 3. Inoculation Chamber 4. Incubator 5. pH Meter 6. Nephelometer.

Laboratory Instrument # 1. Class-Wares:

1. Culture tubes:

Common test tubes in which are used corning or Pyrex glass tubes, are recommended because they have to overcome the effect of high temperatures during sterilization. Culture tubes are used to contain media before and after sterilization. They are also used for making media slopes (slants) for culturing and preserving microorganisms.

2. Cragie tubes (Fig. 16.3):

These are small, thin, open-ended tubes cut from u piece of glass tubing. They are especially used to study bacteria.

3. Durham’s tubes (Fig. 16.4):

Durham’s tubes are small and thin tubes which are generally inserted in an inverted fashion at the bottom of culture containing tubes to detect gas production during fermentation processes.

4. Flasks:

Almost all size of flasks ranging from 50 ml to 1000 ml (1 litre) are generally used for microbial studies in laboratories. Kolle flasks and Roux bottles, which are different from the usual one (Frenbach flask), are sometimes used to help increase the exposure of the medium to the atmosphere. These glass wares are used to contain media before and after sterilization; are also used for culturing micro-organisms in liquid medium.

5. Inoculating loops:

A measured length of platinum wire, nichrome or Eureka wire is fixed into a metal or glass rod at one end and bent suitably at the other. The open part of the wire including the loop is approximately 8 cm in length. Two types of loops can be used.

(i) Smaller loop:

This loop (Fig. 16.5A) is bent in the line with the main axis; the diameter of the loop be around 3.0 nm. These loops are used to inoculate small quantities of inoculum from liquid or solid media.

(ii) Larger loop:

This loop (Fig. 16.5B) is bent at right angle to the main axis; the diameter of the loop be around 5.0 mm. These loops are used to inoculate larger quantities of inoculum and may also be used in direct transfer of microbial colonies from one medium to the other.

6. Inoculating needle:

It is a straight wire fixed at one end of the rod and the other end is straight without any loop (Fig. 16.5C). The inoculating needle is used for making stab culture.

7. Pasteur pipettes:

Pasteur pipettes (Fig. 16.6) have capillary ends and generally range up to 20 cm in length with a wide bore length of 11 cm and a narrow bore length of 9 cm. They are used to transfer small quantities of liquid media.

8. Petri dishes:

The petri-dish (often called Petri plate) was invented in 1887 by a German bacteriologist, namely, Petri. Up to this time Knock had used photographic slides or any other flat piece of glass, which were subject to considerable contamination.

Petri dishes are ‘covered glass dish’ consisting of a base and a lid. Though there are various sizes of Petri dishes, the size of 10 cm x 1.5 cm is considered to be the most suitable for class work. Petri dishes are used to provide a flat surface to the melted culture medium when poured onto its base.

Laboratory Instrument # 2. Colony Counter:

1. Quebec colony counter (Fig. 16.7A):

It is considered to be one of the simplest colony counters generally used in small laboratories. In this, a Petri dish having colonies of microorganisms developed on a solid medium is mounted on a platform. When the Petri dish is illuminated from beneath, the visible colonies can be counted by a lens having magnification of X1.5.

2. Electrical colony counter (Fig. 16.7B):

Highly improved electrical colony counter may also be used to count colonies. It is provided with an electrode to mark the location of each colony counted on a plate. Each colony touched by the electrode is-recorded automatically in the counter.

Laboratory Instrument # 3. Inoculation Chamber:

Several experiments, e.g., transferring cultures from one medium to another for sub-culting and isolation are performed many a time in the microbiological laboratories. For all these purposes, maintenance of aspectic condition is necessary otherwise unwanted microorganisms contaminate the materials.

An equipment called inoculation chamber (Fig. 16.8) permits these operations under aseptic conditions.

1. Construction of inoculation chamber:

Inoculation chamber is a cabinet possessing four doors. Of these, two doors are on the opposite sides and they can be used to place the cultures, etc. into the chamber.

The front panel has two circular doors which allow the insertion of the hands of the operator into the chamber to conduct inoculation, transfer of culture, etc. The cabinet on top is provided with an UV lamp as well as an ordinary tube light. The UV lamp is used for sterilizing the internal environment while the tube light provides illumination if and when required.

2. Procedure for use:

(i) The interior of inoculation chamber is cleaned using a suitable disinfectant such as 70% alcohol.

(ii) The materials required for experimentation inside the chamber are kept through the doors present on the opposite sides of the chamber.

(iii) The UV light is switched on for about 15 minutes to sterilize the interior of the chamber.

(iv) Transfer of materials to conduct inoculation, etc. can be carried out after this period.

(v) It is preferable to keep a spirit lamp which can be used for flaming the inoculation needles or loops before and after inoculation.

Laboratory Instrument # 4. Incubator:

An incubator (Fig. 16.9) is an equipment that consists of copper/steel chamber, around which warm water or air circulates either by electricity or by means of small gas flame. The temperature of the incubator is maintained constant by thermostat control.

Principle:

Incubator is operated to culture or for growing microorganisms in a suitable medium at proper temperature. In an incubator the variation of temperature should not be more than one degree Celsius (1°C). In large incubator the variation of temperature goes up to 2 or 3°C.

Small square type incubators are better than large incubators. If a lower temperature than that of the room temperature is needed, the water, before circulating around the upper chamber, is directed by the thermostat to pass through an ice chest or a small boiler according to the requirement of temperature.

Precautions:

The door of the incubator should be opened only when necessary. If the tubes are to be incubated for a long time or at higher temperature, the medium may turn to be too dry due to excessive evaporation.

In such case, cotton plug should be pushed inside the neck of the tube and a media rubber cap should be placed to cover the plug. If the Petri dishes are to be incubated for a long time these may be placed in moist chamber with a damp sterile cotton wool at the bottom.

Uses:

The method of incubation of culture depends upon the temperature and the oxygen requirements of the microorganism. For this purpose the incubator is used to maintain different temperature required for growth of microorganism in a microbiological laboratory.

Laboratory Instrument # 5. pH Meter:

The pH meter is used to determine the pH (acidity or alkalinity of a solution) of solutions of unknown pH as well as for setting of pH of various media used for the cultivation and testing biochemical activities of microorganisms. In many microbiological experiments, the culture medium must be of a specific type with reference to alkalinity or acidity. This is necessary to culture specific microorganisms.

Similarly during enzymatic-studies, the enzymes are to be harvested and studied in a particular medium with a specific pH, otherwise it (enzyme) will be inactive. When buffer solutions are prepared care is taken to see that they have a specific pH and the pH is checked with the help of an instrument called pH meter. We will first understand what is pH and its importance before studying the working of a pH meter.

1. What is pH?

The hydrogen ion concentration of any solution is called the pH; the pH is expressed as a number from 0 to 14. The number of pH refers to an expression of the concentration of hydrogen ions (H⁺) in the solution—the higher the concentration the more acidic the solution.

However, pH is defined as the negative logarithm of the hydrogen ion concentration. Since pH is equal to the negative logarithm of the hydrogen ion concentration, then

pH = – log 10″⁷

pH = -log

pH = 7

Pure, water then has a pH of 7 and is considered neutral. Values of pH below 7 are acidic and above 7 are basic. Growth and survival of microorganisms is greatly influenced by the pH of the environment and all organisms differ as to their requirements. The specific range for bacteria is between 4 and 9 with the optimum being 6.5 to 7.5. Fungi prefer an acidic environment with optimum being 4 to 6.

A solution with a pH of 8 has hydrogen ion concentration ten times less than a solution with a pH of 7, i.e., its hydrogen ion concentration is 0.00000001 or 10^-8. The values always differ by a factor of 10. A chart of pH values is given as follows (Table 16.2).

2. Measurement of pH with pH meter:

The measurement of pH with pH meter is performed electrometrically and is dependent upon the development of a membrane potential by a glass electrode. The glass electrode (Fig. 16.10) possesses an internal sealed tube fitted with a metallic tip (typically of silver-silver chloride) and an external tube filled with a standard solution. A pH sensitive glass bulb forms the immersion tip of the electrode.

The potential of the glass electrode is proportional to the pH of the solution in which it is immersed. In addition to the glass electrode, a pH meter is provided with another electrode, the reference electrode. The only purpose of this electrode is to complete the measuring circuit with a device that is not sensitive to any of the ions in the solution.

The reference electrode consists of a metallic internal element typically of mercury-mercurous chloride (calomel) or silver-silver chloride immersed in an electrolyte, usually a saturated solution of potassium chloride.

The function of electrolyte is to form a conductive salt-bridge between the metallic element and the sample solution in which the two electrodes are placed. To keep stable electrical communication between the internal metallic element and the sample solution, a liquid junction is present in the tip of the outer body of the reference electrode.

This junction possesses an extremely small hole through which electrolyte solution streams continuously pass into the solution to be measured. (This contaminates measured solution, but the effect is insignificant except with very small volumes.)

The pH meter is fitted with a temperature-compensation circuit for introducing a known potential to balance out the potential caused by different sample temperatures. The instrument also consists of a standardizing potential, which is used to balance the circuit to indicate the correct pH of the standard used as a reference to measure the pH of a sample solution.

3. Precautions taken during use of pH meter:

(i) Glass electrodes when used for the first time should be dipped in distilled water or 0.1 mol/HCl solution for several hours before use.

(ii) The solution should be thoroughly stirred before measuring the pH.

(iii) The temperature of the solution should be kept constant as pH is heat sensitive.

(iv) After every measurement, the glass electrode must be thoroughly washed with distilled water.

(v) The electrodes should not be allowed to dry; they must always be immersed in distilled water.

(vi) Before use, the instrument should be calibrated with a standard solution (a solution of known pH).

Laboratory Instrument # 6. Nephelometer:

Nephelometer is an instrument used for analysing the concentration of a substance in a liquid. The functioning of this instrument lies upon the varying intensity of the light as it passes through the liquid.

The working of nephelometer is based on the measurement of the intensity of the scattered light (light rays that get deflected at right angles to the direct rays as they pass through a medium) as a function of the concentration of the dispersed phase.

Minute particles in the medium undergo scattering to produce a symmetrical pattern of secondary rays with a maximum intensity at 90°, i.e., at right angles to the direct beam (which does not undergo deflection). Since nephelometry is involved in the analysis of deflected rays, the measurement are always done at 90° (secondary rays).

In Nephelometry, measurements are made depending on the comparison between the intensity of light scattered by the sample and the light scattered by a standard suspension under identical conditions. Higher intensity of the scattered light as it passes through the medium indicates higher turbidity, i.e., increased concentration of the sample.

The standard (reference) turbidity indicator used in most nephelometric measurements is a Formazin polymer compound. This compound is preferably used due to its easily dispersible nature in water and uniform light scattering properties when mixed with clay and other natural turbid materials.

The instrument essentially consists of a light source, a sample tube to hold the turbid suspension, and a photocell. The light source is a tungsten lamp, the light rays from which are focussed on to the sample tube holding the suspension to be analysed.

As the light passes through the suspension, two kinds of light rays are produced: direct rays and deflected or scattered rays. Direct rays are absorbed by a light shield and they are not measured by nephelometer.

The scattered rays, which undergo scattering (by the suspension) to the extent of 90° are sensed by a photocell which is kept at right angles to the sample tube. The amount of scattered light that comes out of the sample suspension is directly proportional to the concentration of the sample compound.

Measurement of Turbidity:

1. Reagents:

The following reagents are needed for measurement of turbidity of a solution:

(i) Turbidity free water:

Highly purified distilled water, which can be obtained by passing distilled water through a membrane filter with pore size not exceeding 0.2 micrometer. The water must possess turbidity not more than 0.02 NTU (Nephelo turbidity unit).

(ii) Standard turbidity suspension (stock solution):

The stock solution is prepared with formazin particles with strength of 400 NTU.

The preparation of the stock solution is the following:

(a) Solution A:

One gram of hydrazine sulphate [(NH₂)₂ H₂SO₄] is dissolved in distilled water and the volume is made to 100 ml in a volumetric flask.

(b) Solution B:

10 grams of hexamethylene tetramine [(CH₂)₆ N₄] is dissolved in distilled water and the volume is made to 100 ml in a volumetric flask.

(c) Standard turbidity suspension (stock solution) preparation:

Standard turbidity suspension (stock solution) is prepared as follows:

5 ml of solution A and 5 ml of solution B are mixed in a 100 ml volumetric flask and the mixture is allowed to stand for 48 hours at room temperature. The solution is diluted with distilled water up to 100 ml (up to the mark in the flask).

This suspension will have a turbidity of 400 NTU and it can be used for 4-6 months. To start the procedure, 25 ml of stock solution is taken and diluted with distilled water to make the volume to 100 ml. This suspension possesses a strength of 100 NTU.

2. Procedure:

Turbidity readings are taken as follows:

(i) The instrument is switched on and is allowed a warm up for a period of 10-15 minutes.

(ii) The turbidity range (i.e., 0 to 100 NTU) is selected using the selection knob.

(iii) The sample tube with turbidity free distilled water is inserted into the holder and covered with light shield.

(iv) With the set zero control, the meter is adjusted so that it reads ‘0’.

(v) The tube is removed and is replaced with standard turbidity suspension. The control is so adjusted that the matter indicates 100 NTU, the actual strength of the suspension.

(vi) The standard suspension is replaced with the unknown sample and the turbidity is read directly from the meter reading in NTU scale.

In case the turbity of the sample is more than 100, suitable dilutions with distilled water are used and NTU is calculated as follows: