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2.3: Examples of Bacterial Growth Characteristics in Broths, Slants and Plates - Biology

2.3: Examples of Bacterial Growth Characteristics in Broths, Slants and Plates - Biology


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Even on general purpose growth media, bacteria can exhibit characteristic patterns of growth. Staining procedures and metabolic tests must be used for a definitive identification.

Growth Characteristics in Broths

Growth Characteristics on Slants

Growth Characteristics on Plates


BIO 2410: Microbiology

Frequently during the semester you will need to describe bacterial (or fungal) growth observed on slants or Petri plates. It will be useful to learn the terminology used for describing common colony types. The following outline will be helpful for verbally communicating the appearance of observed colonial growth.

1. Form &ndash The form refers to the shape of the colony. These forms represent the most common colony shapes you are likely to encounter.

1a. Size &ndash The size of the colony can be a useful characteristic for identification. The diameter of a representative colony may be measured in millimeters. Tiny colonies are referred to as punctiform.

1b. Surface &ndash How does the surface of the colony appear? Bacterial colonies are frequently shiny and smooth in appearance. Other surface descriptions might be: veined, rough, dull, wrinkled (or shriveled), glistening.

1c. Color &ndash It is important to describe the color or pigment of the colony. Also include descriptive terms for any other relevant optical characteristics such as: opaque, cloudy, translucent, iridescent.

2. Elevation &ndash This describes the &ldquoside view&rdquo of a colony. These are the most common.

3. Margin &ndash The margin or edge of a colony (or any growth) may be an important characteristic in identifying an organism. Several examples are shown below.


Classification of bacterial culture media on the basis of consistency

Solid medium

Solid medium contains agar at a concentration of 1.5-2.0% or some other, mostly inert solidifying agent. Solid medium has a physical structure and allows bacteria to grow in physically informative or useful ways (e.g. as colonies or in streaks). Solid medium is useful for isolating bacteria or for determining the colony characteristics of the isolate.

Semisolid medium

Semisolid medium is prepared with agar at concentrations of 0.5% or less. Semisolid medium has a soft custard-like consistency and is useful for the cultivation of microaerophilic bacteria or for the determination of bacterial motility.

Liquid (Broth) medium

These media contain specific amounts of nutrients but don’t have a trace of gelling agents such as gelatin or agar. Broth medium serves various purposes such as propagation of a large number of organisms, fermentation studies, and various other tests. e.g. sugar fermentation tests, MR-VP broth.


Culture of Microorganisms: 5 Steps

The following points highlight the five main steps for culture of microorganisms. The steps are: 1. Preparation of Media 2. Adjustment of pH of Media 3. Preparation of Stabs and Slants 4. Pouring of Plates 5. Inoculation of Bacteria in Nutrient Slants and Agar Plates.

Step # 1. Preparation of Media:

In preparing a culture medium for any microorganism, the primary goal is to provide a balanced mixture of the required nutrients, at concentrations that will permit good growth. No ingredient should be given in excess because many nutrients become growth inhibitory or toxic as the concentration is raised.

A medium composed entirely of chemically defined nutrients is termed as synthetic medium. One that contains ingredients of unknown chemical composition is termed a complex medium. For different purposes in laboratory the media is used either ‘solid’ or ‘liquid’.

A. Liquid or Broth Media:

Nutrient broth is the basis of most media used in the study of different types of microbes. It is one of the most important liquid media used for bacteriological purposes.

(i) Flask – 1000 ml capacity.

(ii) Graduated cylinder – 500 ml.

(iii) Beef extract, bactopeptone, distilled water.

(iv) Balance and weight box.

(v) pH paper, comparator block, 0.1 (N) NaOH and 0.1 (N) HCl soln.

To make 300ml of nutrient broth: –

0.9 gm. of beef extract and 1.5 gms of bactopeptone are weighed separately and taken in a coni­cal flask. Then 300 ml dist. water is added and the ingredients are mixed thoroughly. The pH is adjust­ed by adding a little alkali (NaOH soln.). The flask is then plugged with cotton wool and autoclaved at 15 lbs. pressure for 15 minutes.

2. Potato-dextrose Broth:

It is a type of semisynthetic medi­um. This is very often used for the growth medium of fungi which grow better in potato-dextrose broth than in the nutrient broth.

Freshly peeled potato – 400 gms. (40%)

(ii) Graduated cylinder – 250 ml.

(iii) Potato, Dextrose and distilled water.

(iv) Other requirements are as in the previous broth preparation.

To make 200 ml of broth:

80 gms of freshly peeled potato and 5 gms of Dextrose are weighed and taken in a conical flask. 200 ml of dist. water is poured into the flask and the ingredients are mixed thoroughly by a glass rod. (Actually the peeled potato is boiled in flask using 100 ml water for 10 min and the extract is taken by decanting). The flask is plugged and autoclaved at 15 lbs. pressure for 15 min.

B. Solid or Agar Medium:

Nutrient agar is an important medium for bacteriological purpose. It is simply nutrient broth solidified by the addition of agar. Due to its solid consistency, this medium serves as a good device for the culture of bacteria on a solid surface.

Bacteriological peptone – 5 gms. (0.5%)

ii) Graduated cylinder – 500 ml.

iii) Beef extract, bactopeptone, agar, and distilled water.

iv) Balance and weight box.

v) pH paper, comparator block 0.1(N) HCl and 0.1 (N) NaOH soln.

To make 500 ml of nutrient agar:

7.5 gm. of powdered agar is weighed and taken in a flask, containing 250 ml of distilled water. The content is then heated in a water-bath to allow the agar to dissolve. In another flask 1.4 gms of beef extract and 2.5 gms of peptone are dissolved in 250 ml of distilled water. The pH is then adjusted and checked with pH paper.

The two solutions are then poured in a 500 ml flask, stirred thoroughly and then heated gently. Then the medium is dispensed in culture tubes and conical flasks (as stock culture medium is autoclaved at 15 lbs. pressure for 15 min after plugging the tubes and the flask).

2. Potato-Dextrose-Agar (P.D.A.):

Potato-dextrose-agar medium is very often used for the culture of fungi. It is simply potato dextrose broth solidified by adding agar.

Freshly peeled potato – 400 gms. (40%)

(ii) Graduated cylinder – 500 ml.

(iii) Beef extract, bactopeptone, agar, and distilled water.

(iv) Balance and weight box.

(v) pH paper, comparator block 0.1(N) HCl and 0.1 (N) NaOH soln.

To make 300 ml of P.D.A.: –

4.5 gms of agar is taken in a flask containing 150 ml of distilled water. The content is heated in a water-bath to dissolve the agar. 120 gms of freshly peeled potato is taken in a flask and 150 ml water is added to it. It is boiled for 10 mins. Then this potato extract is taken and its volume is made up to 150 ml by adding water. To this extract, 7.5 gms of Dextrose is added and thor­oughly mixed.

Now the above two solutions are poured in a 500 ml flask and stirred thoroughly. This medium is dispensed in culture tubes and flasks. The tubes and flasks are plugged (Fig. 2.1.) and autoclaved.

(i) Agar must be liquefied properly before mixing with broth.

(ii) Agar must not be heated too much other­wise it will lose its ability to solidify.

(iii) Dispensing should be done quickly, otherwise the agar will solidify.

A detailed list of the composition of various media is given in the Annexure.

Step # 2. Adjustment of pH of Media:

Principle:

The hydrogen-ion-concentration of culture media is of prime importance for the successful cultivation of bacteria. Some species grow best in acid medium, others in alkaline medium still others prefer substrates neutral in reaction. The H + concentration i.e.

So, for the growth of a particular macro- organism, the medium should have a specific pH. To adjust the pH, acid and alkali are used.

Requirements:

(ii) pH paper and colour standard

(iv) 0.1(N) NaOH and 0.1(N) HCl soln.

Procedure:

The simplest method for determining the pH of a soln. is to use commercially available pH paper which is impregnated with an indicator. The latter gives a colour change over a pH range of 6.4 to 8.2. A strip of pH paper is cut into small pieces and each piece is placed within a well on the comparator block.

With the help of a glass rod a drop of medium is drawn out and put on the piece of pH paper. The resulting colour is compared with the colour standard supplied.

The pH of the medium, if found to be acidic, is brought to the required pH by adding 0.1 (N) NaOH drop-wise and testing with pH paper after thoroughly mixing with a glass rod. Conversely, 0.1 (N) HCl is used to get an acidic pH of the medium.

Precautions:

(i) While neutralizing the broth, acid or alkali should be added drop-wise.

(ii) After adding acid or alkali the medium should be stirred to ensure proper mixing of the acid or alkali added.

(iii) At every step colour reaction should be noted.

Step # 3. Preparation of Stabs and Slants:

Procedure:

In order to prepare stabs, the medium is poured up to 1/2 of the culture tube (about 20 ml), which is then plugged carefully and sterilised in autoclave. After sterilization, the culture tube is kept erect in a test tube stand until the medium solidifies. Then they are collected in a wire-net basket and preserved.

In order to prepare “slants” the medium is taken up to 1/4 of a culture tube (about 7 ml) with the help of a measuring cylinder and funnel. Then the culture tubes are plugged and autoclaved. After sterilisation, before the medium sets, the tubes are sloped on a bench by leaning them against a length of a wooden stick of 1/2″ thickness in such a fashion that the medium does not touch the plug.

The culture tubes are maintained in that position until the medium solidifies. After solidification of the medium, the “slants” are collected in a wire net basket and preserved with a label indicating the date of preparation and the nature of the medium (Fig. 2.2).

Precautions:

(i) Culture tubes should be plugged with non-absorbent cotton.

(ii) It should be noted that during dispensing, the medium does not stick to the sides of the culture tubes.

(iii) During slanting, care should be taken to see that the medium does not touch the plug.

(iv) Stabbing or slanting should be done just before solidification i.e. at around 47°C, otherwise, there will be water inside the slant.

(v) Stabs and slants should not be disturbed before the medium solidifies.

Step # 4. Pouring of Plates:

Principle:

Plate culture consists of an organism growing on a solid medium contained in a petridish. “Pouring of plates” refers to the process of pouring melted nutrient agar into petridishes. By this process a larger surface area is created for the growth of microbes in all directions.

Requirements:

(i) Nutrient agar “stab” or medium in flask.

(ii) Sterilised petridishes.

(iv) Absorbent cotton, rectified spirit, glass marking pencil etc.

Procedure:

Solid medium contained in culture tubes or flasks is melted in a water-bath. Once the agar is melted perfectly, the medium is allowed to cool around 45°C. The working table is cleaned and sterilised with cotton soaked in rectified spirit. Hands are also sterilised with rectified spirit.

The cotton plug of the melted tube or flask is opened near a flame and the mouth of the tube/flask is flamed in a semi-horizontal position. With the left hand the lid of a petridish is raised far enough to permit the mouth of the tube or flask to enter without touching the sides.

About 15 ml medium is quickly and carefully poured into the petridish, the tube/flask is withdrawn and the cover or lid is replaced. The petridish is tilted slightly with the movement of the wrist to allow homogeneous spreading of the medium (Fig. 2.3). When the medium solidifies, the plates are incubated at 30°C in an inverted position i.e. upside down so that moisture drop does not fall on the medium.

Precautions:

(i) All work should be done aseptically.

(ii) After solidification the plates should be kept inverted.

(iii) During pouring it should be noted that the mouth of the tube/flask does not touch any part of the petridish.

Step # 5. Inoculation of Bacteria in Nutrient Slants and Agar Plates:

Materials Required:

(i) Nutrient slants and nutrient agar plates.

(iv) Bacterial culture slant:

Method:

First the inoculating needle (Fig. 2.4) is sterilised by heating strongly in a flame and cooled by holding it outside the flame. After cooling the needle further by touching it on the ‘extra’ medium (where there is no growth) in supplied slants, a very small amount of inoculum is taken out by means of the needle and then inoculates in the previously prepared slants in a zigzag manner.

Each bacterial culture is inoculated in duplicate. The needle is then flamed to ensure killing of bacteria in the needle. (Fig. 2.5).

For streaking plates, inoculum is taken out in the same way as mentioned and touched on the solid medium in a plate. The needle is flamed to kill excess bacteria and cooled. The inoculum is spread by streaking a line with the needle (Fig. 2.6). The needle is again burnt for the same purpose and cooled.

Continuous and zigzag lines arc streaked so as to reduce gradually the number of cells along the line and to get isolated single colonies after incubation. The whole operation of inoculation is performed aseptically in front of a strong flame. The inoculated slants and plates (in inverted position) are incubated overnight at 37°C.


Factors affecting growth

Agar density vs. nutrient density

Agar is a polymer made of polysaccharide agarose and agaropectin. It is used in bacteria labs as the gelling agent in media for agar plates. Growing bacteria on gel allows for researchers to collect individual colonies much more easily than if they were grown in a liquid medium. The hardness of a gel medium increases along with it agar concentration, and bacteria move more slowly through hard gel. As agar density increases, the width of branches found in fractal patterns of bacterial growth is thinner. If the concentration of agar is very high and the solution is very hard, then the bacteria cannot move. The colony grows in one spot, and grows radially when new bacteria are physically pushed from the center. [4]

Bacteria need nutrients to reproduce and move. A higher nutrient concentration allows bacteria to spread more quickly. Growth patterns are compact at high nutrient densities, and become more fractals as nutrient density decreases [4].

High agar concentration and high nutrient concentration result in a colony with compact, concentric rings, and little to no branching [4]. If agar concentration is high but the nutrient level is low, then bacteria must rely on nutrients diffusing towards the colony. The resulting pattern of growth follows the diffusion-limited aggregation model and forms a branching pattern.

A medium that has a high nutrient level and a moderate concentration of agar will cause bacteria to form a pattern of concentric rings originating from the center. These rings are formed by alternating migration phases, in which the colony moves rapidly, and consolidation phases, during which the colony does not grow. [4]

Diffusion-limited aggregation

Diffusion-limited aggregation (DLA) is a growth model used to predict bacterial growth. It creates complex, multi-branched forms, and can be applied to any system where diffusion is the main method of particle transportation. DLA can be observed in bacterial growth on agar plates, in dendrites, dust balls, electrodeposition, and mineral deposits. [9]

A DLA pattern begins with a seed molecule at the origin of the lattice. A "random walker" molecule diffuses from far away in a random pattern of motion. It stops once it reaches a space adjacent to the seed molecule, and another random walker is launched. In a DLA lattice, a molecule that sticks out of a main branch will be emphasized by new growth, not be rounded or smoothed over. Nodes are more likely to catch wandering particles because they three facets available for growth, compared to a molecule in the branch, which only has one available facet. [5]

Communicating walker model

The communicating walker model is a variation on DLA used to explain how bacteria expand the boundary of their wetting fluid to move into previously unoccupied areas. In this model, the random walker is a particle made up of 1,000–100,000 bacterial cells located on the surface of the media. The walker's metabolic state is fueled by nutrients from the media, and is used to drive bacterial activities and metabolic processes. In a high concentration of nutrients, the internal energy increases until the walker divides, spreading bacterial growth. If there is not enough food for necessary activities, the internal energy of the walker drops to zero, and it is immobile. When active, walkers move in a random pattern, but are confined within the boundaries of the wetting fluid. If the walker attempts to make a movement that would put it outside the wetting fluid, the movement is not performed, but a count is added to that segment of the wetting fluid. Once the number of attempts on that segment reaches a certain threshold, the envelope is pushed out into the new area. The threshold bacteria need to meet to propagate the envelope is directly related to agar concentration, as harder agar requires more attempts to breach [2].

Chiral patterns in bacterial colonies can be explained using the random walker model and assigning each walker an orientation to represent cellular orientation. After every step, the walker rotates to a new orientation according to its assignment and takes a step forwards or backwards [2].

Other factors

Length of cells and handedness of flagella also affect how patterns form. Specifically, they both contribute to chiral patterns of growth. Chirality is observed when bacteria form swirling, hurricane-like patterns on an agar plate. Cells that grow in a chiral pattern are longer than cells that grow in splitting, branched patterns, though the mechanism is not yet understood [1].

Chirality relates to symmetry, and a chiral patterns are partially a result of flagella "handedness." If a bacterium's flagella favor movement to the right or to the left, it cause will the colony's pattern to go in either a clockwise or counterclockwise direction [1].


Intracellular Bacteria

  • Those that can be cultured in microbiologic media in the laboratory (facultative) or
  • Those that required living cells/animals (obligate).

Facultative Intracellular Bacteria

  • Legionella pneumophila: It prefers the intracellular environment of macrophages for growth. Legionella induces its own uptake and blocks lysosomal fusion by an undefined mechanism.destroys the phagosomal membrane with which the lysosomes fuse.
  • Mycobacterium tuberculosis: M.tuberculosissurvives intracellularly by inhibiting phagosome-lysosome fusion.
  • Listeria monocyotogenes: Listeria quickly escapes the phagosome into the cytoplasm before phagosome-lysosome fusion.: Very resistant to intracellular killing by phagocytic cells.

Obligate intracellular bacteria

This group of bacteria can’t live outside the host cells. For e.g. Chlamydial cells are unable to carry out energy metabolism and lack many biosynthetic pathways, therefore they are entirely dependent on the host cell to supply them with ATP and other intermediates. Because of this dependency Chlamydiae were earlier thought to be a virus.

All viruses are obligate intracellular parasites.

Obligate intracellular bacteria cannot be grown in artificial media (agar plates/broths) in laboratories but requires viable eukaryotic host cells (eg. cell culture, embryonated eggs, susceptible animals).

    cannot be cultured in vitro it is an obligate intracellular parasite.
  1. Coxiella burnetti: The metabolic activity of Coxiella burnettiiis greatly increased in the acidic environment of the phagolysosome.
  2. Ricekettsia spp

Toxoplasma, Cryptosporidium, Plasmodium, Leishmania, Babesia, and Trypanosoma are obligate intracellular parasites.


Aseptic Technique

A. Different objects will increase in their level of contamination with decreased temperature.

B. Different object will have the same level of contamination.

C. Environments in contact with animals and humans are likely to be less contaminated due to hygiene practices.

A. This range is close to human body temperature, a temperature preferred by many of these organisms

B. This range denatures enzymes that make these organisms pathogenic.

C. The range is energy efficient.

A. The mouth is held in the flame while the inoculating tool is inserted for removal of organisms

B. The cap is removed and placed on the table away from the flame

C. The bottom of the tube is help in the flame for 10 seconds

A. The introduction of a known organism into a culture

B. The introduction of an unwanted organism into a culture

A. Before the inoculum is picked up from the culture tube

B. Right before introducing inoculum to sterile medium

A. Take lid off and set upside down on bench

B. Open one side of the lid diagonally off the plate

1. To heat the bottom of the sterile slant tube before adding sample

2. To heat the loop/needle right after obtaining sample from slant culture

3. To flame the mouth of the slant culture tube after removing cap and before replacing the cap

4. To flame the mouth of the sterile slant tube after removing cap and before replacing the cap

5. To heat the loop/needle right before obtaining the sample from slant culture


TYPES OF CULTURE MEDIA

Culture media (singular: medium) are nutrient medium or preparations that supports and allow microorganisms to be propagated in the laboratory for further study. They are artificial growth medium that support the growth of microbes outside their natural host or environment. Culture media provide all necessary nutrients and growth factors that encourage the development of the organism inoculated in it. Microorganisms are usually introduced into the culture media (which can be solid, liquid or semi-solid) through a process known as inoculation, and the inoculated plates or medium are incubated at optimum temperature and later observed for microbial growth known as culture.

A culture in microbiology refers to colonies of microorganisms (bacteria in particular) that grow and multiplies in or on a culture medium. Artificial microbiological culture media provide all the essential environmental and nutritional requirement of the organism(s) to be culture because virtually all pathogenic microorganisms and some commensals are chemoorganoheterotrophic in their mode of nutrition and rarely produce their own food. Peptone, yeast extract, meat extract, water and agar re some of the main constituents of most microbiological artificial culture media.

There are several culture media available for the propagation of microorganisms as well as for the transportation and storage of microbes in the microbiology laboratory. The choice of which type of culture media to use is often influenced by many factors including but not limited to the nutritional requirement of the microorganism, the natural habitat of the microbe and on the experience or particular need of the scientists. Table 1 shows some of the basic culture media commonly used for various bacteriological and mycological investigations in the microbiology laboratory.

Table 1. Illustration of some microbiological culture media

Microbiological culture media especially those for bacteriological and mycological studies are classified into different categories and these shall be highlighted in this section.

  • GENERAL PURPOSE MEDIA:General purpose media or basic medium is a routine culture media which is used for the cultivation of microorganisms in the microbiology laboratory. General purpose media can also be called simple or basal culture media. They are basically used for the culturing of bacteria that do not need extra growth nutrients, and are not fastidious in nature. General purpose media support the growth of a wide variety of bacteria, and they do not contain any growth inhibitory substance. Commonly used general purpose media in the microbiology laboratory include nutrient agar, nutrient broth, peptone water, tryptic soy broth, tryptic soy agar, Mueller-Hinton broth and Mueller-Hinton agar amongst others. Mueller-Hinton (MH) agar is the best medium for conducting antimicrobial susceptibility testing (AST) in the microbiology laboratory (Figure 1).
Figure 1. Illustration of Mueller-Hinton agar plates inoculated with test bacteria and single antibiotic disks prior to incubation for antimicrobial susceptibility testing (AST). Photo courtesy: https://www.microbiologyclass.com
  • ENRICHMENT AND ENRICHED MEDIA:Enrichment media are usually liquid media or broth that supports the growth of a particular bacterium while inhibiting the growth of unwanted bacteria. Typical example of an enrichment medium is the Selenite F broth culture medium which is used for the culture of faecal specimens. Selenite F broth inhibit the growth of commensals or non-clinically relevant bacteria in faecal specimens prior to their subculture onto solid culture media plates. Alkaline peptone water is another example of an enrichment medium and enrichment media are generally used to recover pathogens from faecal samples. Enriched media are culture media that also contain additional growth nutrients (e.g. blood, serum and egg yolk) like enrichment media for the cultivation and isolation of fastidious bacteria but unlike enrichment media, enriched media are mainly solid culture media and they are generally used to cultivate fastidious bacteria e.g. Streptococcus species and Haemophilus species. Blood agar and chocolate agar are examples of enriched culture media.
  • SELECTIVE MEDIA:Selective media are culture media that promote the growth of certain type of bacteria while inhibiting the growth of the undesired organisms. Such culture media contain inhibitory substances such as dyes, salts and antibiotics which prevent the growth of undesired microorganisms by suppressing them so that only the desired microbes will grow. Selective media used in the microbiology laboratory for the culture of microbes include mannitol salt agar (which contain NaCl that inhibit some bacteria), MacConkey agar (which contain bile salts and crystal violet that inhibit the growth of Gram-positive bacteria), Sabouraud dextrose agar (which contain antibiotics that inhibit bacterial growth) and Tellurite media (which contain potassium tellurite that inhibit many bacteria excluding Corynebacterium diphtheria). Another example of a selective medium used for bacteriological investigation is the Lowenstein-Jensen media (that contains egg yolk) used for the isolation of Mycobacterium tuberculosis from mixed cultures or specimens. Sabouraud dextrose agar (SDA) and Salmonella-Shigella agar (SSA) are selective media used for the cultivation and isolation of fungi and enteric pathogens (e.g. Shigella and Salmonella) respectively. SDA is selective because it contains cycloheximide and chloramphenicol which inhibit the growth of saprophytic fungi and bacteria respectively while allowing only the fungi of interest to grow.
  • DIFFERENTIAL MEDIA:Differential media are growth media that allow certain bacteria to have distinct colonies on the culture media. Organisms growing on differential media produce characteristic colonies that differentiate them from other group of microbes. They are also known as indicator media because they contain certain indicators (e.g. neutral red, chemicals and dyes) which changes in colour especially when the definite organism (i.e. the organism of interest) is present in the specimen being cultured. Unlike selective media which only encourage the growth of particular microbes, differential media differentiate between different groups of bacteria and some culture media can serve as both selective and differential media. Differential media or indicator media are also used for the presumptive identification of some bacteria. For example, MacConkey agar (MAC) is a differential media that help microbiologists to differentiate lactose fermenting bacteria (e.g. Escherichia coli) which ferments lactose to produce pink colonies on MAC from non-lactose fermenting bacteria (e.g. Salmonella) which does not ferment lactose and thus appear as pale or colourless colonies on the growth medium.Examples of differential media are cystein lactose electrolyte deficient (CLED) media, mannitol salt agar (MSA), MacConkey agar and blood agar amongst others.
  • TRANSPORT MEDIA:Transport media are used for transporting specimens or microorganisms from one location to another. They are mainly used in cases where the samples collected will not be cultured immediately after collection. Transport media provide all the nutrient and environmental factors necessary to preserve the samples and/or organisms en route to the laboratory where the formal investigation will take place. Of most importance is the fact that transport media prevent the overgrowth of commensals or contaminants in the collected samples Examples of transport media include Amies medium and Cary-Blair media.
  • STORAGE MEDIA:Storage media which may include a basic or general purpose media such as nutrient agar in slants or tubes are used to preserve and store microbial cultures for long period until they are needed for formal or further laboratory investigations. Chalk cooked meat broth and egg saline medium are typical examples of storage media used to store bacterial cultures in the microbiology laboratory. There are several reasons of storing or preserving microorganisms and maintaining them for many days, weeks, months or years in the laboratory under controlled conditions before reviving them again for futuristic use. Microorganisms including bacteria, fungi, protozoa and viruses can be preserved in different ways and in different types of media in order to study them in the future or to use them for research. Microbes are preserved for epidemiological, microbiological, educational, and clinical purposes especially for the diagnoses of infectious diseases. Microorganisms can also be preserved for commercial purposes – in which unique strains of microbes are maintained in dormant but viable states to be sold for research purposes and other industrial usages, which are all geared towards enhancing research works in the field of microbiology. There are several organizations that specialize in the storage of microorganisms for commercial purposes and these companies serve as sources for obtaining important strains of microbes that guide microbiological research in the industry, hospitals and in educational institutions. The American Type Culture Collection (ATCC) and the National Collection of Type Culture (NCTC) are typical examples of such companies that maintain microbes in a dormant but viable state for commercial purposes and other research purposes too. One of the simplest method of maintaining the viability of microorganisms especially bacteria in the microbiology laboratory is by a periodic subculturing of the bacteria onto freshly prepared culture media preferably in nutrient agar slants made in McCartney or Bijou bottles. In this crude technique of preserving microbes in the microbiology laboratory, bacteria to be preserved is subcultured from a culture media plate onto the slant of a nutrient agar bottle and the organism is subcultured onto freshly prepared nutrient agar slants on a weekly or monthly basis – while ensuring that other environmental conditions such as temperature and pressure are maintained at optimal levels for the growth of the organism being preserved. However, this method though cheap and less cumbersome, may give room for mutation to occur in the organism being subcultured at intervals. To counter this problem, there is several commercial maintenance or microbial preserving culture media in the open market that allows microbiologists to preserve their cultures over certain periods of time. These processes as described above only preserve microbes for short-term purposes. For long-term preservation of microbes, the process of lyophilization (freeze-drying) is highly recommended for the preservation of microorganisms and other important laboratory working materials for many years. The concept of lyophilization or freeze-drying is used for the long-term storage of microorganisms and it removes volatile substances such as water from the material being stored and the preserved material is held under high vacuum in freeze-drying liquids like liquid nitrogen. Bacterial cultures are maintained at a very low temperature and in a dry state using the technique of lyophilization. When a culture is required for any microbiological purpose after freeze-drying, it is simply reconstituted with nutrient broth or distilled water – which resuscitate the dormant but viable organism to start growing again.

Further reading

Brooks G.F., Butel J.S and Morse S.A (2004). Medical Microbiology, 23 rd edition. McGraw Hill Publishers. USA.

Goldman E and Green L.H (2008). Practical Handbook of Microbiology, Second Edition. CRC Press, Taylor and Francis Group, USA.

Madigan M.T., Martinko J.M., Dunlap P.V and Clark D.P (2009). Brock Biology of Microorganisms, 12 th edition. Pearson Benjamin Cummings Inc, USA.

Mahon C. R, Lehman D.C and Manuselis G (2011). Textbook of Diagnostic Microbiology. Fourth edition. Saunders Publishers, USA.

Patrick R. Murray, Ellen Jo Baron, James H. Jorgensen, Marie Louise Landry, Michael A. Pfaller (2007). Manual of Clinical Microbiology, 9th ed.: American Society for Microbiology.

Wilson B. A, Salyers A.A, Whitt D.D and Winkler M.E (2011). Bacterial Pathogenesis: A molecular Approach. Third edition. American Society of Microbiology Press, USA.

Woods GL and Washington JA (1995). The Clinician and the Microbiology Laboratory. Mandell GL, Bennett JE, Dolin R (eds): Principles and Practice of Infectious Diseases. 4th ed. Churchill Livingstone, New York.


Watch the video: How to Properly Inoculate from Broth to Broth, Broth to Slant, and Broth to Plate TSB, NAS, NAP (May 2022).


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