In this article we will discuss about the three categories of methods in order to obtain particular kind of organism, physical, chemical and biological methods.
1. Physical Conditions for Growth:
(i) Heat Treatment:
This is applied in case of bacteria which form spores for example Bacillus spp. Lactobacillus spp. etc. To select the endospore-forming bacteria, a mixed culture can be heated at 80°C for 10 min before being used to inoculate culture medium. In such case spore forming bacteria will survive and all the bacteria (vegetative cells) will be killed.
(ii) Incubation Temperature:
Most of the microorganisms require a particular temperature to grow efficiently. Such a temperature is called optimum temperature. To select psychrophilic or psychrotrophic bacteria, cultures are incubated at extreme low temperature varying zero to 5°C. The mesophihc (20-40°C) and thermophilic (45°C and above) microbes will be killed, and, only psychrophiles will survive.
(iii) pH:
It is an important factor which influences the growth of microorganisms. Most of the lactobacilli spp. grow at acidic range of pH (below 7) while some bacteria grow at alkaline medium e.g. Vibrio cholerae.
(iv) Cell Size and Motility:
Most of the microorganisms has a definite size and are motile in nature. Due to 0.15 mm size Treponema can be isolated by placing gingival scrapping on the surface of an agar plate. It will penetrate from the filter and will swim through solid agar medium. The hazy appearance in the agar indicates the presence of Treponema.
2. Chemical Methods:
The chemicals select a particular microorganism. If it is supplemented in a medium only those microorganisms will grow well which can utilize the supplemented chemicals. This particular kind of selection is based on addition of either carbon or nitrogen source. Such method is often called enrichment culture and medium is called enrichment medium.
In addition certain dyes such as rose bengal are often used for the isolation of fast growing fungi, while certain antibiotics such as nystatin is added to avoid bacterial contaminants in the medium. Similarly, yeast mannitol agar (YEM) medium supplemented with congo red allows to differentiate rhizobia with that of Agrobacterium.
Rhizobia do not take colour while Agrobacterium colonies become pink. Many Gram-positive bacteria do not grow in the presence of dyes due to their inhibitory action while Gram-negative bacteria grow well in the presence of dyes.
3. Biological Methods (Natural Selection):
The presence of particular microorganisms is specific in a habitat. Certain microorganisms would like to live in dung only (coprophilous fungi and methanogenic bacteria), while others live in citrus fruit e.g. penicillia.
Nature itself selects the microorganism. Streptococcus pneumoniae in sputum sample is contaminated by many other organisms. However, laboratory mice are extremely susceptible to infection by S. pneumoniae and if the sputum sample is injected into lab mice, the pathogen will multiply rapidly and rest of the microorganisms will be killed due to defensive mechanism of animal.
I. Methods of Culturing Aerobic Bacteria:
Aerobic or facultative bacteria are to be grown in an atmosphere rich in oxygen. For this a large surface of medium in such condition increases the exposure. There are several devices such as Fembach flask, kolle flask and Roux bottle which are used for providing increased aeration (Fig. 3.1)
II. Methods of Culturing Anaerobes:
The anaerobic cultures differ in their requirements and sensitivity to oxygen. Some bacteria such as Clostridium histolyticum is aero-tolerant and may show growth on the surface of anaerobic culture plates, while others such as C. tetani is strictly anaerobic and form surface growth if the oxygen is less than 2mm Hg.
The obligate anaerobic forms can be grown in anaerobic growth cabins where oxygen is replaced by inert gas (Helium or CO2). Such chambers or cabins are expensive. Hence, a less expensive device is the use of ‘candle jar’. The inoculated cultures of the Petri dishes are placed in the jar, a candle placed on the top of the Petri dishes is lit, and the jar is sealed with an air tight lid.
As the candle bums indicating the utilization of oxygen and production of carbon dioxide, which supports bacterial growth. The anaerobes grow in agar stab cultures, whereas facultative anaerobes are able to grow in either the absence or presence of the oxygen.
The microtolerants or microaerophiles are those which grow in the presence of low oxygen concentration. Their growth stops in the presence of high oxygen concentration (Fig. 3.2)
Now-a-days commercial anerobic jar are available which has an indicator strip which change the colour from blue to colourless showed total consumption of oxygen (Fig. 3.3).
Use of ‘Gas Pak’ for Preparing Anaerobic Jar:
The gas pak is commercially available as a disposable envelope, containing chemicals which generate hydrogen and CO2 on the addition of water. After the inoculated plates are kept on the jar, the gas pak envelope with H2O added is placed inside and lid screwed tight.
Hydrogen and carbon dioxide are liberated and the presence of cold catalyst in the envelope permits the combination of hydrogen and oxygen to produce an aerobic environment.
The gas pak is simple and effective, eliminating the need for drawing a vacuum and addition of hydrogen. The reduction of oxygen in the medium is achieved by the use of various reducing agents such as 7% glucose, 1% thioglycolate, 0.1% ascorbic acid, 0.05% cysteine.
III. Culture Characteristics:
1. Colony Appearance:
Many fungi produce colonies with a fluffy appearance similar to cotton wool. The molds produce colonies which on aging develop a dry chalky appearance. The colony characteristics are given in Fig. 3.6.
2. Colony Forms:
The colony shape may be circular, filamentous, rhizoidal, punctiform (dot like), irregular or spindle shape.
3. Colony Elevation:
This form is used to describe the depth of the colony developed by microbes. A colony may be flat (thin film over the agar surface), raised, convex or umbonate or with papillate surface.
4. Colony Margins:
The margins may be entire, undulate (wavy), crenate, dentate, lobate, rhizoidal or filamentous (Fig. 3.6).
5. Optical Density:
The colony may be transparent or translucent (foggy in appearance) or opaque (not permitting light to pass through) or iridescent (rainbow colour).
6. Colour:
Many microbes develop colonies which are pigmented. Such coloured substances are either water soluble or insoluble. The soluble pigments diffuse out in the medium.
7. Colony Odour:
Some microbes produce a characteristic smell which sometimes helps in identifying the microbe. The actinomycetes produce an earthy odour which is quite often experienced after a first shower or rain. Many fungi produce fruity smell while E. coli produces a faecal odour.
8. Colony Consistency:
The degree of thickness, solidity or firmness of the medium should be examined as given below:
(a) If the entire broth appears milky and cloudy, it is called turbid.
(b) If a deposit of cells is present at the bottom of the tube, the term sediment is used.
(c) When microbial or bacterial growth forms a continuous or interrupted sheet over the broth, it is called pellicle.
(d) If the growth of microbe is similar in appearance to that of butter, it is called butyrous.