laboratory diagnosis of bacteria

laboratory diagnosis of bacteria
The basic flow of procedures involved in the laboratory diagnosis of infectious disease is as follows :
- Direct examination of patient for the presence of agents.
- Growth & cultivation of the agents from these same specimens.
- Analysis of the cultivated organisms to established their identification & other characteristics.

Microscope is defined as the use of it to magnify (visually enlarge) objects too small to be visualized with naked eye so that their characteristics are readily observable because most infectious agents cannot be detected with naked eye.







































Growth medium
A growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms or cells. There are different types of media for growing different types of cells There are two major types of growth media: those used for cell culture, which use specific cell types derived from plants or animals, and microbiological culture, which are used for growing microorganisms, such as bacteria or yeast.
The most common growth media for microorganisms are nutrient broths and agar plates; Some organisms, termed fastidious organisms, require specialized environments due to complex nutritional requirements. Viruses, for example, are obligate intracellular parasites and require a growth medium containing living cells.
Types of culture media according to their composition:
1-Solid media
2-Liquid media
3- Semi solid media are often mixed with agar at percentage (0.3-0.5)% (a carbohydrate substance obtained from (Sea Weeds) dissolved in 90-100 degree , solidified at 32-42 degree & remain liquid at 45-50 degree .

Types of culture media according to their nature
1- Nutrient media
a source of amino acids and nitrogen (e.g., beef, yeast extract)
** This is an undefined medium because the amino acid source contains a variety of compounds with the exact composition being unknown. Nutrient media contain all the elements that most bacteria need for growth and are non-selective , its also known as a basal or complex medium that contains:
-a carbon source such as glucose for bacterial growth
-water
-various salts needed for bacterial growth
**A defined medium (also known as chemically defined medium or synthetic medium) is a medium in which
-all the chemicals used are known
-no yeast, animal or plant tissue is present

2-Minimal media
Minimal media are those that contain the minimum nutrients possible for colony growth, generally without the presence of amino acids, its contain
-a carbon source for bacterial growth, which may be a sugar such as glucose.
-various salts, which may vary among bacteria species and growing conditions; these generally provide essential elements such as magnesium, nitrogen, phosphorus, and sulfur to allow the bacteria to synthesize protein and nucleic acid
-water .

3-Selective media
Selective media are used for the growth of only selected microorganisms. For example, if a microorganism is resistant to a certain antibiotic, such as ampicillin or tetracycline, then that antibiotic can be added to the medium in order to prevent other cells, which do not possess the resistance, from growing. examples of it
· eosin-methylene blue agar (EMB) that contains methylene blue – toxic to Gram-positive bacteria, allowing only the growth of Gram negative bacteria
· YM (yeast and mold) which has a low pH, deterring bacterial growth
· MacConkey agar for Gram-negative bacteria
· Hektoen enteric agar (HE) which is selective for Gram-negative bacteria
· mannitol salt agar (MSA) which is selective for Gram-positive bacteria and differential for mannitol
4-Differential media
Differential media or indicator media distinguish one microorganism type from another growing on the same media. This type of media uses the biochemical characteristics of a microorganism growing in the presence of specific nutrients or indicators (such as neutral red, phenol red, eosin y, or methylene blue) added to the medium to visibly indicate the defining characteristics of a microorganism. examples of it:
· blood agar (used in strep tests), which contains bovine heart blood that becomes transparent in the presence of hemolytic Streptococcus
· eosin methylene blue (EMB), which is differential for lactose and sucrose fermentation
· MacConkey (MCK), which is differential for lactose fermentation
· mannitol salt agar (MSA), which is differential for mannitol fermentation
5-Transport media
Transport media should fulfill the following criteria:
· temporary storage of specimens being transported to the laboratory for cultivation.
· maintain the viability of all organisms in the specimen without altering their concentration.
· contain only buffers and salt.
· lack of carbon, nitrogen, and organic growth factors so as to prevent microbial multiplication.
· transport media used in the isolation of anaerobes must be free of molecular oxygen.
· example of it Thioglycolate broth for strict anaerobes.
6-Enriched media
Enriched media contain the nutrients required to support the growth of a wide variety of organisms, including some of the more fastidious ones. They are commonly used to harvest as many different types of microbes as are present in the specimen. Blood agar is an enriched medium in which nutritionally rich whole blood supplements the basic nutrients. Chocolate agar is enriched with heat-treated blood (40-45°C), which turns brown and gives the medium the color for which it is named.

7-Assay Media
This media contain the chemical substance like vitamins , amino acids & antibiotic example of it Muller Hinton medium which used for determine the antibiotic sensitivity.

8-Enumeration Media
This media which used to determine the amont & the number of of microorganisms in liquid like milk & water example of it Plate count agar.

9-Maintenance Media
This media which used to maintenance the bacterial activity for long peroid of time example of it Nutrient broth with 10% glycerol. This media would be kept at low temperature about -70 degree.

Inoculation Methods
1- Streaking Method on solid media by using the loop .
2- Spreading Method on solid media by using the loop , Spreader or the swap.
3- Stap Method on semisolid media by using the needle or by spreading the growth on the surface of the slant by using the loop.

· Inoculation from liquid to liquid media occur by the use of loop or swap.
· Inoculation from liquid to solid media occur by the use of loop or swap.
· Inoculation from the specmain to solid or liquid media occur by the use of swap.
· Streaking Method are impartment in order to obtain the single colony to be culture on the other media which selective to that organism. .
· When completed the inoculation methods we cultivate the plates in the incubator at 37degree for 24 hr ( some organisms may be grow at 42 or 25 degrees for 3-5 days ) then either we used the growth immediately or storage in the refrigerator for one week then we put it in autoclave in order to remove it .