ENUMERATON AND IDENTIFICATION OF BACTERIA ON USED HANDKERCHIEFS IN MALES


ENUMERATION AND IDENTIFICATION OF BACTERIA ON USED HANDKERCHIEFS IN MALES

ABSTRACT

The enumeration, identification and characterization of bacteria on used handkerchiefs in males was carried out.  A total of forty (40) handkerchiefs were collected across the levels from the male students of Faculty of Pharmacy, University of Benin.

The handkerchiefs were soaked in a sterile water and the specimen (bacteria) were cultured on nutrient agar, blood agar, and MacConkey agar for the enumeration, identification and recovery of bacteria present.

For enumeration, 37 cultures yield countable growths and 3 cultures yielded no growth.  For identification and characterization, the positive growth was identified and characterized using standard protocols.  One out of forty (40) cultures yielded serratia species, sixteen cultures yielded Streptococcal species, nine cultures yielded Staphylococcus epidermidis, eight cultures yielded Staphylococcus aureus, and three cultures yielded staphylococcus saprophyticus.

On the whole, the bacteria, showed different growths in different media and as well different reactions to different biochemical tests.

CHAPTER ONE

1.0 INTRODUCTION

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.

Identification of Gram Negative Rods

Table 1a: Common Microbiota of the Respiratory Tract and Skin

ORGANISM GRAM-STAINED CELL MORPHOLOGY COLONY MORPHOLOGY,ETC

Haemophilus influenzae Very small gram-negative coccobacilli with capsules in young cultures.  Older cultures show longer rods and pleomorphic forms, and lose the capsule.  Non-motile. Small, round, convex, translucent, oxidase-negative colonies appear only on Chocolate agar (prefer 10% CO2) OR as satellite colonies on blood agar, around hemolytic S. aureus colonies.  Requires “X” & “V” factors (hemin and NAD or NADP).

Haemophilus spp. Small gram-negative rods, short or long chains, often pleomorphic Small, smooth, opaque colonies.  Most require X and/or V factors from blood-based enriched medium.

Gram-negative enteric rods e.g. Klebsiella positive). Gram-negative rods, usually larger than above spp. Usually large, gray, glistening colonies.  Will grow on MacConkey’s. 

Gram-netative rods, non-fermentative e.g. Alcaligenes, Acinetobacter. Gram-negative bacilli or coccobacilli. Small, gray, glistening colonies.  No reaction in TSI butt.

Micrococcus luteus Gram-positive cocci, usually in tetras. Lemon-colored colony.  Common flora of skin.

Corynebacterium spp. (diphtheroids) Gram-positive, pleomorphic rods, some club-shaped; bipolar or barred stain; groups form “palisades”. Varies with species.  Usually smooth, convex, whitish, opaque, non-hemolytic.  Some wrinkled. 

1.1 NORMAL FLORA OF THE SKIN

The majority of the bacteria found on the skin are in the most superficial layer of the epidermis and upper part of the hair follicles.  They are generally non-pathogenic and are considered to be commensals although multialistic and parasitic roles have been assigned to them. For example, skin bacteria or their metabolite on the skin may protect against colonization by dermatophytic fungi.  The predominant resident micro-organisms of the skin are acrobic and anacrobic diphtheroid bacilli (e.g., Corynebacterium, propionibacterum), non-haemolytic acrobic and anacrobic staphylococci (Staphylococcus epidermidis, occasionally staphylococcus aureus); Gram-positive acrobic spore-forming bacilli that are ubiquitous in air, water and soil; alpha haemolytic streptococci (streptococcus viridians) and enterococci (strephtococci faecalis); and Gram-negative coliform bacilli and Acinetobacter.

Non-pathogenic mycobacteria occur in areas rich in sebaceous gland secretions (e.g. external ear.).

1.2 NORMAL FLORA OF THE RESPIRATORY TRACT

The nasal cavity is the gateway to the respiratory tract.  It acts as a route of entry of bacteria into the respiratory tract.  This respiratory tract can be subdivided anatomically into:

1. The upper airways consisting of the anterior and posterior nares and nasopharynx.

2. The middle airways made up of the oropharynx and tonsils.

3. The lower airways with pharynx, trachea, bronchi and lungs.

This classification serves as a useful tool for examining the dynamics of the airways colonization.  The structural and physiological differences at each site provide an environment compatible for some bacteria and hostile to others (Barlows, 1998).

The bacteria normally found in the nares (nostrils) are mainly the staphylococcal organisms (staphylococcus epidermidis and staphylococcus aureus), streptococcal organisms (streptococcus pneumonia), Neisseria organisms (Neisseria meningitides), enterobacteriacae, protest species, Haemophillus influenzae, lactobacillus species, corgnebacteria and mycobacteria (Toders, 2002).

The nostrils are always heavily colonized predominantly with staphylococcus epidermidis and corynebacteria with staphylococcus aureus which is mainly found in the nostrils (Prescott, 2002).  A large number of bacterial species colonize the upper respiratory tract.  The common organisms include the non-haemolytic and alpha-haemolytic streptococci and neisseria but sometimes pathogens such as streptococcus preumonia, streptococcus pyogenes.  Haemophilus influenzae and neisseria meningitides colonize the pharynx (Caugnant, 1994).

Other micro-organisms found in the nose include corynebacterium, peptostreptococcus and fusobacterium.  One rare occasions streptococcus viridans can be isolated from the nasal canals.  In healthy individuals, streptococcus pyogenes and streptococcus pneumonia have also been isolated from the nostrils.

The lower respiratory tract (trachea, bronchi, pulmonary tissues) is virtually free of micro-organisms mainly because of the efficient cleansing action of the cilliated epithelium which lines the tract.  Any bacteria reaching the lower respiratory tract are swept upward by coughing, sneezing etc.

Therefore, the respiratory tract is colonized by a large number of resident microbial flora that varies as a result of change in exogenous and endogenous condition (Todars, 2002).

Table 1b:  MICROBIAL FLORA OF THE NASAL CAVITY 

(Todars, 2002)

Micro organisms Occurrence

Staphylococcus epidermidis

Staphylococcus aureus *

Streptococcus pneumonia *

Neisseria species

Neisseria Meningitidis *

Corynebacteria ++

+

+/-

+

+

++

Proteus species

Haemophilus influenzae *

Mycobacteria +

+

+/-

++=Nearly 100%

+=Common

+/-=Rare

*=Potential pathogen

1.3 NORMAL FLORA OF THE HUMAN ORAL CAVITY

The presence of nutrients, epithelial debris and secretion makes the mouth a favourable habitat for a great varieties of bacteria.  Bacteria found in the oral cavity include streptococci, lactobacilli, staphylococci and corynebacteria, with a great number of anbacrobes, especially bacteroides.  The normal flora occupies available colonization sites which makes it more difficult for other micro-organisms to become established.  Indigenous oral flora contributes to host nutrition through the synthesis of vitamins, and to the host immunity by inducing low levels of circulating and secretory antibodies that may cross react with pathogens.

Oral bacteria exert microbial antagonism against certain pathogens by production of fatty acids, peroxides, bacteriocins etc which inhibit or kill non-indigenous species.

1.4 NORMAL FLORA OF THE CONJUCTIVA

A variety of bacteria may be cultivated from the normal conjuctiva but the number is usually small.  Staphylococcus epidermidis and certain corynforms (propionibacterium acne) are dominant.

Staphylococcus aureus, some streptococci, haemophilus species and Neisseria species are occasionally found.  The conjuctiva is kept moist and healthy by the continuous secretion by the lachrymal glands.  Blinking wipes the conjuctiva every few seconds while mechanical washing of the conjuctiva with water washes away foreign objects including bacteria.

Lachrymal secretions also contain bactericidal substances including lysozymes.  There is little or no opportunity for micro-organisms to colonize the conjuctiva without, species mechanisms to attach to the epithelial surfaces and some ability to withstand attack by lysozymes.

1.5 CHARACTERIZATION OF THE NORMAL FLORA OF THE BODY

a. Staphylococcus aureus:  It is a Gram positive cocci that grow in large, round, opaque colonies at 37oC.  It is a facultative, non-sporing anacrobe and can withstand high salt concentration (7.5 – 10), extreme pH, high temperature (60oC for 1 hour) and can remain viable for months.  It is pathogenic due to the presence of exotoxins.  It produces catalase enzymes and it ferments carbohydrates.

b. Staphylococcus epidermidis:  They have the same characteristics as staphylococcus aureus but are different because they are non-pigmented, do not produce coagulase and are less pathogenic.

c. Neisseria species:  They are shaped and paired with their flat sides touching.  They are acrobic or microphillic organisms with an oxidative form of metabolism.  They produce catalase enzyme for sugar fermentation, cytochrome oxidase enzymes and possess pilli.  They are strict parasites and do not survive long outside their host.  Capsules are found in pathogenic strain (Neisseria meningitides and Neisseria gonorrhea).

d. Corynebacteria:  They are Gram positive irregular non-spore forming rods.  They are acrobic and are capable of producing catalase and posses mycolic acids and unique type of peptidoglycan on the cell wall.

e. Mycobacteria:  These are fast bacilli with a complex layered structure composed of high molecular weight of mycolic acids and waxes.  This makes them acid fast bacteria.  They are long slander straight or curved rods with a tendercy to be filamentous.  They are strict acrobes and grow well on simple nutrient medium.  Some colonies are pigmented while others are not.  They are non-sporing and non-motile.

f. Escherichia coli:  It is rarely found on the nasal cavity of the body.  It is a coliform bacterium.  It is the most common acrobic non-fastidious Gram negative rod.  It is capable of fermenting lactose reducing nitrates to nitrites and it is oxidase negative.  They are non-motile.

g. Haemophillus influenzae:  They are tiny Gram negative pleomorphic rods fastidious and sensitive to drying, temperature extremes and disinfectant.  They grow best on blood agar, they are facultative anacrobes and do not produce oxidase.  They are non-motile.

h. Streptococcus pneumonia:  They occur as a lancet-shaped cells arranged in pairs.  They are Gram positive cocci requiring blood or chocolate agar.  It is an alpha-haemolytic and produces smooth or micoid colonies.  They are non-motile, non-sporing, non-catalase producing facultative anacrobes.  The pathogenic strains are smooth and possess capsule while the non-pathogenic strains are rough and have capsules.  They are also referred to as pneumococcus and were formerly classified as Diplococcus pneumonia until their genetic similarity to streptococci was demonstrated.

These bacteria are capable of attaching to host cells or tissue surfaces by means of ligards or adhension.  Example can be seen below in table 1c.

Table 1c: Examples of specific attachment of bacteria to host cell or tissue surfaces (Todars, 2002).

Bacterium Bacteria ligard for attachment Host cell or tissue receptor Attachment Site

Streptococcus pneumoniae Cell-bround protein N-acetythexamine galactose disaccharide. Mucosal epithelium

Staphylococcus aures Cell-bround protein Amino terminus of filronectin Mucosal epithelium

Mycoplasma Membrane protein Sialic acid Respiratory epithelium

1.6 BENEFITS OF NORMAL FLORA

The presence of these bacteria in human body has certain benefits as listed below:

1. The normal flora prevents colonization by pathogens by competing for attachment sites or for essential nutrients.  This is thought to be their most beneficial effect.

2. The normal flora may antagonize other bacteria through the production of substances which inhibit or kill non-indigenous species.

3. They synthesize and excrete vitamin in excess of their own needs which are absorbed by their hosts.  For example, enteric bacteria secrete vitamin k1.

4. They stimulate the development of some certain tissues such as the caecum and certain hymphatic tissue in the gastrointestinal tract.  The caecum of germ free amonials is enlarged, thin walled, and fluid filled.

5. The normal flora stimulates the production of cross reactive antibodies.  Normal flora act like antigens in humans.  Thus, they induce immunological responses in particular antibody mediated immune (AMI) response.  Low levels of antibodies produced against components of the normal flora are known to cross react with certain related pathogens and thereby prevent infection or invasion.  These antibodies produced against antigenic components of the normal flora are sometimes referred to as natural antibodies and are lacking in germ-free animals.

1.7 FACTORS AFFECTING THE MAKE-UP OF NORMAL FLORA 

(Todars, 2002)

1. AGE:  In childhood, the make-up of the normal flora is difficult to establish due to poorly developed immune systems.  In old age, the immune system degenerates.  However, in adulthood, the systems are fully matured and functional.

2. SEX:  Some organisms are gender determined.  Example, syphilis caused by Treponema palladium in women and gonorrhea caused by Neisseria gonorrhea in men.

3. STRESS:  Stress decreases the efficiency of the immune system and this leads to an alteration in the make-up of the normal flora causing the object to be prone to infection by the bacteria.

4. NUTRITION AND DIET:  A good diet ensures that an individual is healthy and the normal flora is not compromised.

However, it should be noted that some of these flora found in the body are either transient (that is, they are acquired by contact) or resident.

Moreso, these bacteria commonly found on the body may cause infections such as:

1. Nasal vestibulities caused by staphylococcus.  This is an infection of the nasal vestibules.

2. Follicuditis which is inflammation of hair follicles in the skin.  Stephylocucci are also implicated in this infection.

3. Furuncles characterized by the presence of boils on the inflamed area of skin containing pus.  This infection is usually caused by staphylococcus aureus entering through the hair follide. 

1.8 ANTIBACTERIAL AGENTS USED IN THE TREATMENT OF INFECTIONS CAUSED BY BACTERIA FOUND ON USED HANDKERCHIEFS

Most of the bacteria found on used handkerchiefs in males are pathogenic and causes disease to the individual using the handkerchief.  Therefore, antimicrobial agents are required for the treatment of these infections.

An antimicrobial agent is a substance which could be of natural, synthetic or semi-synthetic origin which at low concentration kills or inhibit the growth of micro organisms (www.surgery encyclopedia.com).  Antimicrobial agents could be antibacterial, antifungal or antiprotozoan agents.

An antibiotic was originally defined as a substance produced by a micro-organism which inhibits the growth of other organisms (Hugo, 1988).  The discovery of synthetic methods has resulted in a modification of the definition above.

An antibiotic now refers to a substance produced by a micro-organisms or to a similar substance (produced wholly or partly by chemical synthesis) which in low concentration inhibits the growth of other micro organisms.

Chemotherapy is the use of chemical substances for the treatment of diseases.  These chemicals substances are referred to as chemotherapeutic agents.  Any chemical used in the treatment, relief, or prophylaxis of disease is defined as a chemotherapeutic agent.  It must possess the property of selective toxicity that is; it kills or inhibits the growth of micro organisms but causes little or no harm to the host.

1.9 MODE OF ACTION OF ANTIBACTERIAL AGENTS IN CLINICAL USE (BARLOWS, 1998).

Antibacterial agents exhibit a wide range of actions.  One of the ways to classify them is according to their site of action in the bacterial cell wall.  Examples include:

⦁ Agents acting on the bacterial cell wall synthesis.

⦁ Agents acting on the bacterial protein synthesis.

⦁ Agents acting on nucleic acid synthesis.

⦁ Agents acting on bacterial cell membrane.

The classification of antibacterial agents according to their site of action in the bacterial cell is shown below:

Cell Wall Protein Synthesis Nucleic acid Cell membrane

Synthesis synthesis

Penicillins Aminoglycosides Sulphonamide Polymixins

Cephalospomis Chloramphenicol Diaminopyrimidine Gramicidin

Other β-lactams Tetracyclines Quinotones Pyrocidin

Bacitracin Macrolides Rifamycin Moneusin

CycloserinFusidic acidNitroimidazoles

IsoniazidStreptomycinNitrofurams

MupironNovobiocin

1.10 AIMS AND OBJECTIVES

⦁ To enumerate the number of bacteria on used handkerchiefs in male.

⦁ To identify and characterize the bacteria present on used handkerchiefs in males.

.

ENUMERATON AND IDENTIFICATION OF BACTERIA ON USED HANDKERCHIEFS IN MALES



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