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Micro-organisms are ubiquitous and are found in almost every area around human bodies.  Some are specifically found in certain regions of the body as a normal flora where they live as commensals with man.  This association is important in protecting the body against other infectious diseases.
Each area of the body surface acquires a characteristic flora of organisms well adapted to growth at that particular environment.  These residents (normal flora) tend to suppress the intruders either by competition for space and food supply or by production of metabolites that are antagonistic to the survival of the intruder.
These residents could be dislodged from their environment when sneezing, coughing, belching, yawning or could be destroyed by regular use of antiseptic soaps or creams on the body surfaces.
Handkerchiefs often used in males for wiping face, closing of the mouth and nose when expressing these reflex activities, therefore constitute an abode for bacteria.  Furthermore, bacteria found in handkerchiefs could differ from one individual to another as the bacteria found could be a reflective of the environment and pathological conditions of the individual using the handkerchief.  For instance, individual with upper respiratory tract infection are likely to dislodge strains of pathogenic microbes along sides with the normal flora in these regions.
Enumeration of bacteria on used handkerchief in males can be done using microscopic cell count and viable cell counting.  Microscopic counts can be done on either samples dried on slides or samples in liquid.  A viable cell counting is the one that is able to divide and form offspring.
Viable cell counting is also called plate count and there are at least two ways of performing plate count: the spread plate and pour plate method.
In spread plate method, a volume of appropriately diluted culture is spread over the surface of an agar plate using a sterile glass spreader.  The plate is then incubated until colonies appear, and the number of colonies formed are counted.
In pour plate method, a known volume of culture is pipetted in a sterile petridish plate.  Molten agar medium is then added and mix well by gentle swirling of the plate on the bench top.  Because the sample is mixed with molten agar medium, the bacteria to be counted must be able to withstand brief temperature exposure to the temperature of the molten agar (45 – 50oC).  Here, the colonies formed are counted throughout the plate and not just on the agar surface as in the spread plate method.
In identifying bacteria, the morphological and biochemical characteristics of the bacteria, are evaluated.  The appearance and the microscopic description of the bacteria are examined with the aid of a light compound microscope.
From the growth of the bacteria (pure culture), the specimen to be viewed under the microscope can be prepared as a smear or as a wet mount.  A stain is used to contrast the specimen from the background.  This strain could be basic or acid stain.  Basic stain, example methylene blue and crystal violet, are cationic and have a positive charge.  They are ideal for staining chromosomes and the cell membrane of the bacterial.  The acid stains are anionic and have a negative charge, and are used to stain cytoplasmic material and organelles or inclusions.  Common examples are eosin and picric acid.
There are two types of stains – simple and differential.  A simple stain has a single basic dye that is used to show shapes of cells and structures within a cell while a differential stain consists of two or more dyes and is used in the procedures to identify bacterial.  One of the most commonly used differential stain the gram stain.  Gram positive bacteria stain purple while Gram negative bacteria stain pink.  Other biochemical tests like Indole test, Urease test, Catalase reaction, Oxidase reaction etc will help in further characterization of the bacteria identified from the handkerchief.
            Identification of Gram-Positive Cocci 
Gram-positive cocci can be identified by the growth on blood and chocolate agar and the catalase test.  The catalase test is used to differentiate those bacteria that produce the enzymes catalase, such as staphylococcus from non-catalase producing bacteria such as streptococci.  For catalase positive, gram positive cocci, coagulase test could be used to further differentiate them.  Staphylococcus aureus is coagulase positive whereas Staphylococcus epidermidis is negative to coagulase test.  Streptococci are catalase-negative gram-positive cocci and are further classified on the basis of their type of haemolysis, A (partial) and B (complete) haemolysis on blood agar, Alpha-haemolytic streptococci, streptococca viridians and Streptococoal pneumonia can be differentiated by the optochin disc susceptibility test.  The B-haemolytic streptococci are group according to the lancefield classification.
Identification of Gram-Negative Cocci
Members of this group can be identified using fermentation patterns.  In addition, catalase and oxidase tests can also be performed.  It can further be identified by growing them on Thayer-Martin medium and nutrient agar.

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