Pure culture: A culture that contains only one kind of microorganism is called a pure culture. A culture which contains more than one kind of microorganism is called a mixed culture. A pure culture has only a single type of colony. Pure cultures are essential in order to study colony characteristics, biochemical properties, morphology, staining reaction, immunological reaction or susceptibility towards antimicrobial agents of particular strain of fungus, bacteria or actinomyces.Colony characteristics is defined in the form of colony appearance, colony elevation, colony margin, optical density, color, colony odor and colony consistency. Biochemical properties are based on the reaction carried out inside cell. This include different carbohydrate utilization, protein utilization, lipid utilization and other metabolic reaction such as catalase, oxidase etc.
Morphology indicates shape and size of bacteria such as rods, cocci, spirilli and pleomorphic. Among them, some are long rod, short rod, large cocci, bunch cocci, streptococci and short cocci. Staining reaction helps to determine whether the organism is gram positive or gram negative, capsulated or non-capsulated, spore producer or non-spore producer.
Immunological reaction helps to differentiate the organism on the basis of serological reaction which is based on the different types of antigens present in microorganism. Some organism are susceptible to antibiotics whereas some are resistant to them. On the basis of antibiotic sensitivity, we can distinguish the particular type of organism. A pure culture always show some properties at any time unless mutation occur.
Like all other organism, microorganism also require basic nutrients for survival. The food material which is fed to microorganism for their growth in lab is known as culture medium and the growth is called culture. Although all microorganism have same basic requirements but there is a diversity in using organic or inorganic compounds. The use of culture media vary in form depending on species of microorganism to be used. Some media contains solution of inorganic salts and may be supplemented with one or more organic compounds. Other media may contain complex ingredients such as extracts or digest of plant and animal tissue. Two broad classes of culture media are used in microbiology.
1. Chemically defined (synthetic media)
2. Chemically undefined (natural or empirical, non-synthetic media)
Chemically defined media: These are prepared by adding exact amounts of highly purified inorganic or organic compounds to distilled water. Therefore, the exact chemical composition of defined medium is known. These are needed for the growth for autotrophs (nitrosomonas, nitrobacter etc.) and are used for defining the nutritional requirement of heterotrophs.
Chemically undefined media: It employs digest of casein (milk protein), beef, soybeans, yest cells or any of a number of other highly nutritious compound (yet chemically undefined) and used for culturing heterotrophs. However, there are some media whose chemical composition is partially known and are also used for culturing heterotrophs. Example; potato dextrose agar, mac-conkey agar.
Agar: agar is the main agent used for solidifying the culture media. It liquefies on heating at 96°C and hardens into a jelly on cooling to 40 – 45°C is used to solidify liquid media. On the basis of consistency, which demonstrate the physical characteristics of the media. It has been divided into 3 types.
1. Solid or synthetic medium
2. Semisolid or floppy agar medium
3. Liquid broth
Solid or synthetic medium: When 1.5 % to 2 % agar or sometime gelatin is added, liquid broth becomes solid. Such media used for making agar-slants or slopes. These media are commonly used for isolation and enumeration.
Semi solid or floppy agar: These media are prepared by adding 0.5 % agar. This type of may be selective, which promotes the growth of one organism and retard the growth of another type of organism.
Liquid or broth: In such cases, no agar is added. After inoculation of cells, the growth of cells is visible in form of mass on the top called pellicle. This medium is used for rapid growth of microorganisms.
Type of media on the basis of different purposes
1. Simple media/ general purpose media/ basal media:
It favors the growth of all type of organism and routinely used for diagnostic purpose in laboratory. Example; nutrient agar, nutrient broth. It is also used for growing many non-fastidious bacteria including Escherichia coli, Bacillus species, Staphylococcus aureus, and Pseudomonas species.
2. Special media:
In this media, special compounds are used for growing particular type of organism. These are further classified as following.
a. Enriched media: When some special nutrients such as blood or serum, egg or meat pieces are added to basal media, the latter is called enriched media. It is required for fastidious type of microorganism.
Egg yolk agar ——– Mycobacterium tuberculosis
Blood agar ——– Haemophilus influenza
b. Enrichment media: It is always a liquid (semi-solid) media that flavors the multiplication of particular species of bacteria by incorporating special compounds which selectively favor its growth and inhibits its competitor
Alkaline peptone ——– Vibrio cholera
Selenite – F- Broth ——– Salmonella species
c. Selective media: These media provide nutrient that enhance the growth and predominance of particular type of bacterium and do not enhance other type of organism that may be present on that media. For instance, a medium in which cellulose is only the carbon source will specially select or enrich the growth of cellulose utilizing organisms when it is inoculated with a soil sample containing many kind of bacteria.
Thayer Martin agar ——– Neisseria gonorrhea (contains antibiotics that do not affect N. gonorrhea but inhibit the growth of contaminating bacteria.
Chopped meat glucose agar ——– favors only to anaerobes (contains cysteine amino acid which is reducing agent)
Mannitol Salt agar ——– contains salt which is favorable for salt loving pathogenic Staph. aureus bacteria
Tellurite Blood agar ——– Corynebacterium diphtheria
Mac-Conkey agar ——– favors gram negative bacteria
d. Differential media: It contains reagents and supplement. When incorporated into culture, it allows differentiation of various kind of bacteria. For example; if a mixture of bacteria is inoculated into blood containing agar media some of the bacteria may cause hemolysis of red blood cells but others do not. Thus one can distinguish between hemolytic and non-hemolytic on the same media.
Eosin Methylene Blue agar ——– differentiates E.coli and Aerobacter aerogens
Mac-Conkey agar ——– differentiates lactose fermenting and non-lactose fermenting bacteria
e. Sugar media: The standard sugar media is used for biochemical test contained 1 % sugar concentrated in peptone water along with an indicator. A small tube (Durham’s tube) is kept inverted into the sugar tube containing media is used for detecting gas production.
Hiss Serum agar ——– Pneumococcus sp.
f. Transport maintenance media: Such media are used for transportation of specimen. When specimens example feces, throat swab, urine, pus, nasal swab, urethral discharge are to be sent to lab from remote places, pathogenic bacteria may not survive due to overgrowth of other species of
Glycerol saline media ——– Dysentery bacilli
Name : Pratiksha Shrestha
Ms. Shrestha holds masters degree in food engineering and bioprocess technology from Asian Institute of Technology (AIT) Thailand. She is currently working for Government of Nepal at Department of Food Technology and Quality Control (DFTQC), Kathmandu. She is also a teaching faculty in College of Applied food and Dairy Technology (CAFODAT) affiliated to Purbanchal university, Nepal.