CHAPTER ONEINTRODUCTION1.1S.I.W.E. S Overview

The Students’ Industrial Work Experience Scheme (S.I.W.E.S) is a planned and supervised training intervention based on stated, specific learning and career objectives, and geared towards developing the occupational competencies of the participants. S.I.W.E.S was established by I.T.F in 1973 to solve the problem of lack of adequate practical skills preparatory for employment in industries by Nigerian graduates of tertiary institutions and it helps to provide each student the opportunity for practical and work experience outside the university through attachment to establishments / companies. It helps to compliment the theoretical knowledge gained by students in school.

In its near four decades of existence, the I.T.F has not only raised training consciousness in the economy, but has also helped in generating a corps of skilled indigenous manpower which has been manning and managing various sectors of the Nigerian economy. The scheme exposes students to industry based skills necessary for a smooth transition from the classroom to the industrial and labour market. It affords students of tertiary institutions the opportunity of becoming familiar with and exposed to the needed experience in handling some machinery and equipment which may not be available in their various institution of learning.

It has been proven that learning can effectively be achieved through both classroom teaching and practical demonstration (both of which every prospective graduate must be well versed in). A truly productive individual must not only be knowledgeable but must also be versatile in the application of the acquired skills.

S.I.W.E.S as one of the I.T.F programmes has been designed to give Nigerian students studying technical, experimental and industrial related courses in tertiary institutions the experience that would supplement their theoretical understanding. Consequently, it has become a necessary pre-requisite for the award of diplomas and degree certificates in such courses. It has also become an innovative phenomenon in human resources development and training in Nigeria.

1.2Aims and Objectives of S.I.W.E.S

The major benefits accruing to students who participate conscientiously in industrial training are the skills and competence they acquire. These Relevant Production Skills (RPSs) remain a part of the recipient’s lifelong assets which cannot be totally divorced from them. This is because the knowledge and skills acquired through the training are internalized and become relevant when the need arises to perform technical or experimental occupation and employment (Mafe, 2009).v The I.T.F’s policy documents No. 1 of 1973 (I.T.F, 1973) which established S.I.W.E.S outlined the objectives of the scheme. The objectives are to:

Provide an avenue for students in institutions of higher learning to acquire industrial skills and experience during their course of study;

Prepare students for industrial work situations that they are likely to meet after graduation;

Expose students to work methods and techniques in handling equipment and machinery that may not be available in their institutions/universities.

Opportunity for students to blend theoretical knowledge acquired in the classroom with practical hands-on application of knowledge required to perform work in industry and/or Relevant Production Skills (R.P.Ss);

Enhance students’ contact for job placement after graduation, advancing the relationship between educational and industrial sectors preparing students for the challenges in working environment before and after graduation

1.3ORGANIZATION AND OPERATION OF S.I.W.E.S

The organization of the Students’ Industrial Work Experience Scheme (S.I.W.E.S) may involve many stakeholders as follows:

Federal Government (Federal Ministry of Commerce and Industry); Industrial Training Fund (S.I.W.E.S Division) Supervising/Regulatory Agencies (NUC, NBTE, NCCE);Industry/Employers (NECA, NACCIMA, MAN, Government Establishments); Tertiary Institutions (Universities, Polytechnics, Colleges of Education) and Student trainees (Engineering, Sciences, Technology, NCE Technical).

S.I.W.E.S is operated as a joint venture through the contributory activities of the stakeholders identified above and as shown. The Federal Government (F.G) funds the scheme through the Federal Ministry of Commerce and Industry (FMC&I). It also lays down broad policies and guidelines that govern the scheme. The Industrial Training Fund (I.T.F), a division of the Federal Ministry of Commerce and Industry (FMC&I) is responsible for all the overall management of the scheme in collaboration with other stakeholders. I.T.F collaborates with all stakeholders directly or indirectly.

1.4 BRIEF HISTORY OF IAR&T

The institute of Agriculture Research and Training (I.A.R&T), Obafemi Awolowo University is a national multi-commodity institute for research, services and training for agricultural development in Nigeria.The pioneer School of Agriculture in Nigeria was established on Moor plantation in 1921.The establishment of the College of Animal Health and Production Technology, Ibadan followed in 1964.The three colleges and the Research Division of the western Region Ministry of Agriculture and Natural Resources became the Institute of Agricultural Research and Training, Ibadan in 1969 following a charter signed by the Vice Chancellor of the university of ife (now Obafemi Awolowo University) and the Governor of the former Western Region of Nigeria. IAR&T Serves the needs of the Nigerian farmers in general and farmers in south-western Nigeria in particular within the context of its integrated agricultural resources development and training strategy. The institute has a lot of staffs which include professors, senior scientists with PhD degrees, and junior scientists with M Sc.degrees, technical, administrative and supporting staff.

ORGANIZATIONAL CHART

(GOVERNING COUNCILOBAFEMI AWOLOWO UNIVERSITYGOVERNING BOARDDIRECTORDEPUTY DIRECTORDEPUTY REGISTRARCHIEF ACCOUNTANTLAND AND WATER RESOURCES MANAGEMENTPROGRAMME CEREAL IMPROVEMENT PROGRAMMELIVESTOCK IMPROVEMENT PROGRAMMEFARMING IMPROVEMENT PROGRAMMESOIL MICROBIOLOGY LABORATORYSOIL PATHOLOGY LABORATORYSOIL BIOCHEMISTRY LABORATORY)

1.4.1

1.4.2 Mission and focus of the institute

To serve as a national centre for integrated improvement of the genetic

potential, yield, utilization and nutritional qualities of major foods and agro-industrial crops as well as livestock breeds adapted to the broad agro-ecological zones of south-western Nigeria. IAR&T investigates, evaluates, develops and promotes Farming System based on improved technologies aimed at increasing and maximizing the overall agricultural productivity and production. IART&T provides relevant junior, intermediate and high level manpower training for National Agricultural Development. The Institute also collaborates with other universities, national, regional and international institutions in the practical application and adoption of improved agriculture production technologies.

1.4.3 The organizations of the institute

IAR&T is heads by a Director, who is assisted by a Deputy Director and Heads of Programmes/Units.

Cereals improvement programme

Develops and selects high yielding, disease or pest resistance or tolerant, adaptable, open pollinated and hybrid maize, in addition to pop corn, sorghum and rice varieties for the major agro ecologies in Nigeria.

Farming Systems Research and Extension Programme

Develops and validates on-farm production technologies that are adaptable to the socio-economic circumstances of the resource- poor farmers in South-Western Nigeria for adoption.

Livestock improvement programme

Focuses attention on improved management and productivity of the small ruminants (sheep and goats), trypanotolerant cattle, pigs, rabbits, poultry and the unconventional livestock e.g snails and grass cutter through breeding and selection for disease resistance or tolerance and adaptation.

Crop production unit

Produces breeders and foundation seeds for the national seed service and other seed producers in Nigeria. The unit tests seeds for mechanical purity and viability and also develops effective methods of breaking seed dormancy.

Land and Water Resources Management Programme

Conducts research on the development of improved soil and water management technologies nationwide. It also provides technical support services through laboratory analysis of soils, rocks, plants, fertilizers and water needed for the formulation of recommendations on appropriate land/soil management practices. My internship was at this section specifically at the soil microbiology laboratory where I did some research on the isolation of microorganisms that can degrade heavy metals and the enzyme activities of the soil.

CHAPTER TWO

2.0 THEORETICAL REVIEW

2.1 LABORATORY

A laboratory is a place, building or a part of a building used for scientific related works that may be hazardous to personnel who are required to work there. The works conducted in a Laboratory may include teaching or learning, research, clinical or diagnostic testing and analysis. A laboratory may have associated areas including preparation, instrumentation, decontamination, wash-up and storage rooms, or a workshop in an engineering area (e.g. mechanical, electrical, aeronautical and civil engineering) a medical or clinical laboratory is a laboratory where tests are usually on specimens to obtain information about the health of a patient as pertaining to the diagnosis, treatment, and prevention of disease. Laboratories are commonly used for scientific disciplines ranging from biology microbiology, biochemistry, chemistry, histology physics, botany and zoology to medicine, psychology, dentistry, engineering, agriculture and veterinary science.

2.1.1 FUNCTIONS OF A LABORATORY

The laboratory plays a key role in improving the health of the community. These roles include the followings:

The investigation of the occurrence of diseases and various health problems e.g bacterial, fungal and viral infections

Assisting the radiologist in determining the severity of a patient’s condition

2.2 LABORATORY SAFETY RULES AND PRECAUTIONS

Laboratory coat should be worn while working in the laboratory.

Hand gloves should be worn when handling clinical samples and specimen in the laboratory. Long hair-do is not allowed in the laboratory; it should be packed or rolled neatly to the back. Always wash your hand before and after carrying out each test and while leaving the laboratory.

Do not eat or drink in the laboratory. Any injury sustained in the laboratory should be reported and treated as soon as possible.

Sharp object like lancet and needle should be disposed into the safety box after usage to avoid direct inoculation.

Ensure proper handling of laboratory equipment and instruments.

Clean/ disinfect work bench and equipment before and after practical

Avoid pipetting with your mouth

Use a nose mask to prevent inhalation of aerosols and gaseous suspension of chemicals

Good disposal of used apparatus such as cover slid should be ensured.

Labeling of specimens and record keeping should be done accurately to avoid errors which may lead to mistaking of identity of patients. Personal belongings must not be left on the working bench.

The use of jewelleries should be minimal in the laboratory, it must not dangle.

2.3 LABORATORY APPARATUS AND USES

MICROSCOPE: It is the basic equipment in the laboratory. It is used to magnify microorganisms since they are invisible with the human eyes.

AUTOCLAVE: It is laboratory equipment used for sterilisation of apparatus and media such as MaCartney bottles, sensitivity medium, blood agar etc. It works at 121 degree centigrade for 15minutes.

CENTRIFUGE: It is used for separating solution or liquids into components based on their weight. The test tubes are arranged properly facing each other with equal spaces in between them to ensure the stability of the machine and specimen and to avoid the damage of the equipment as instability can lead to a severe damage.

INCUBATOR: It is a device used to grow and maintain microbiological cultures like culture media.

DRY OVEN: It is used to dry objects, tools and materials like stained smears and also media.

TEST TUBES: They are used for some biochemical tests and other purposes in the laboratory such as serial dilution, nutrient media e.t.c.

PETRIDISHES: They are used for preparing culture media.

pH METER: this equipment is used measuring the hydrogen ion concentration of a liquid sample.

SPECTROPHOTOMETER: this equipment is used in measuring the amount of growth of microorganisms in a nutrient broth by measuring it turbidity through it absorbance level. The higher the growth the higher the absorbance due to higher turbidity. It is also used for enzyme quantification.

DESICCATOR: This is used in drying samples and reagee, it contains silica gel

LAMINAR FLOW MACHINE: It provides a sterile environment for the inoculation and cultivation of microorganisms from samples.

REFRIGERATOR: This is used in storing sample to prevent or minimize microbial activities of the sample; it is also used to reduce the metabolic activities of microorganisms.

HAND GLOVES, NOSE MASKS, and HAIR NET: These are used to protect the body from contamination when working in the laboratory.

INOCULATING LOOP: This is used for picking inoculums and inoculating.

SWAB STICK: It is used for swabbing microorganisms on solidified agar plate.

FUME CUPBOARD: This equipment is used in carrying out laboratory works aseptically.

SPIRIT LAMP OR BUNSEN BURNER: It is also used in sterilizing the working environment and any equipment used.

MICROSCOPIC SLIDE: It is a small glass that is rectangular in shape. The objects and specimens to be viewed under the microscope are placed on the microscopic slide. It is used for Gram staining, catalase test.

COVER SLIP: It is used to cover prepared samples for microscopy view.

BIJOUX BOTTLE: It is used for serial dilution and peptone water preparation

BURETTE WITH RETORT STAND: It is used in titrating for soil respiration analysis.

WHATMAN FILTER PAPER: This is used in filtrating plant extracts for antimicrobial potency.

STERILE SYRINGES: Used for serial dilution.

CONICAL FLASKS AND BEAKERS: They are used for collecting samples, media and reagents preparation and for titration.

MEASURING CYLINDER: Used for measuring water samples.

FUNNEL: It is used for filtering samples.

3.1 SOIL MICROBIOLOGY LABORATORY

3.1.1 CULTURE MEDIA

Culture media is a solid, liquid or semi-solid designed to support the growth of microorganisms or cells, or small plant like the moss. Different types of media are for growing different type of cells.

The two major types of media are those used for cells culture, which use specific cell type derived from plants or animal and microbiological. A growth medium or culture medium is a solid, liquid or semi-solid designed to support the growth of microorganisms or cells, or small plants like the moss Physcomitrella patens.

Different types of media are used for growing different types of cells.

The two major types of growth media are 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 fungi. The most common growth media for microorganisms are nutrient broths and agar plates; specialized media are sometimes required for microorganism and cell culture growth. 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.

3.1.2 COMPOSITION AND PREPARATION OF CULTURE MEDIA

COMPOSITION OF A CULTURE MEDIUM

· Distilled water

· Peptone

· Meat extracts

· Yeast extracts

· Mineral salts

· Carbohydrates

Preparation of Culture Media

There are various media but the common ones used in the laboratory are in powered form or with different chemical composition and examples of these media are Aleksandiov medium, Pikovskaya’s medium, Jensen’s medium, Nutrient agar, Potato dextrose agar, peptone water etc. The basic steps for preparing these culture media are listed below:

· I measured out accurately the required amount of the powered media into a required quantity of water in a sterile conical flask following the manufacturer's specification and stirred thoroughly until completely dissolved.

· It was heated to bring the powders into solution and stirred while heating to avoid burning.

· After heating, I covered the conical flask with cotton wool and foil paper and allow it to cool. Then sterilized in an autoclave at 121oCfor 15 minutes and allow it to cool.

· I poured inside sterile Petri dish (20ml per plate).

· I allowed to cool and solidified, then dried in the oven to avoid steam being present prior to use.

3.1.3 USES OF CULTURE MEDIA

· Culture media are used to grow, transport and store microorganism

· Culture media are used for selection and identification of microorganism testing of antibiotic sensitivity and analysis of water and food samples.

· Culture media are used to demonstrate the presence of organisms which may be causing diseases, when indicated, to test the sensitivity of pathogens to antimicrobial agents.

3.1.4 TYPES OF CULTURE MEDIA

Liquid media: This is also known as broth. Examples include: peptone water, selenite F. They are used for biochemical testing, blood culture, testing for motility and as enrichment media. The major disadvantage is that purity of the growth cannot be guaranteed.

Solid media: This media is the basis of every other media. When organisms are grown on solid media, they grow and multiply at the site of inoculation and form multiple colonies. A liquid medium is made solid by the incorporation of a solidifying agent which does not alter the nutritional content of the medium.

Advantages of solid media:

(a) Bacteria may be identified by studying the colony character,

(b) Mixed bacteria can be separated. Solid media is used for the isolation of bacteria as pure culture. 'Agar' is most commonly used to prepare solid media. Agar is an ideal solidifying agent as it is:

(a) Bacteriologically inert, i.e. no influence on bacterial growth,

(b) It remains solid at 37°C, and

(c) It is transparent.

3.1.5 CLASSES OF CULTURE MEDIA

GENERAL PURPOSE MEDIA: This is majorly used for the cultivation of various microorganisms example is nutrient agar.

BASAL MEDIA: This is the basis of all media and an example is the nutrient broth and when an enrichment agent is added it supports the growth of microorganisms more.

SELECTIVE MEDIA: They are solid media which contains substances that slow down or inhibit the growth of microorganisms other than those of which the media are devised. Examples of this media includes: Potato Dextrose Agar (PDA) and Saboraud Dextrose Agar (SDA) for the cultivation of fungi, Deoxycholate Citrate Agar (DCA) for the cultivation of stool pathogen such as Escherichia coli and Salmonella- Shigella Agar (SSA) also for the cultivation of stool pathogen such as Salmonella and Shigella.

DIFFERENTIAL MEDIA: This media contains substances or indicators that will differentiate one organism from another either by their colours or colonies of the surrounding medium which is as a result of an indicator present in the medium. Examples of this media include MacConkey Agar which differentiates lactose fermenter from non-lactose fermenter. On MacConkey agar Klebsiella spp and Escherichia coli produces a pinkish colouration and also ferments lactose which differentiates them from other organisms which are non-lactose fermenter and an example is Streptococcus spp. The indicator present in the MacConkey agar is METHYL RED.

NOTE: MacConkey agar is a selective and a differential culture medium designed to selectively isolate gram negative and enteric bacteria (normally found in the intestinal tract) and differentiates them for lactose fermentation. Another example is the blood agar which differentiates haemolytic bacteria from non-haemolytic ones.

ENRICHED MEDIA: they are culture media that are enriched with whole or lysed blood, serum, special extracts or nutrients to support the growth of those bacteria that cannot grow on basal media. Examples of this media are: blood agar, chocolate agar, serum agar etc.

For the preparation of blood agar medium, blood is added at a temperature of 45ºc. It can also be referred as a differential medium because it distinguishes bacteria by their type of haemolysis. The medium contains beef extract, tryptone, NaCl, 5-10% blood (which may be from sheep, cattle or horses), agar (solidifying agent).

For the preparation of chocolate agar medium, blood is added at a temperature of 90ºc to cultivate fastidious pathogens. The high temperature at which the blood is added help to lyses/break the red blood cell. The breakdown of the red blood cells releases the X and V factors to the medium i.e. X in form of iron(haematin) and V in form of protein(Nicotinamide Adenine Dinucleotide; NAD+). Examples of fastidious organism include Streptococcus pnuemoniae, Neisseria spp, Haemophilus spp, Pseudomonas spp.

ENRICHMENT MEDIA: They are similar in function to the selective media. The only difference is that selective media are solid. This medium may contain inhibitory substances that control the growth of competitive organism. Examples of these medium includes selenite F, peptone water et c. Selenite F is used to cultivate and isolate Salmonella para-typhi from faeces, urine, water, food.

3.2 MICROSCOPY

3.2.1 OVERVIEW OF THE MICROSCOPE

Microscopes are the devices designed to enable us to see things that are otherwise too small for our naked eye to observe. In the centuries since their initial development, microscopes have been drastically improvised, and our ability to both magnify and resolve object has increased drastically

There are many types of microscopes, and they may be grouped in different ways. One way is to describe the way the instruments interact with a sample to create images, either by sending a beam of light or electrons to a sample in its optical path, or by scanning across, and a short distance from, the surface of a sample using a probe. The most common microscope (and the first to be invented) is the optical microscope, which uses light to pass through a sample to produce an image. Other major types of microscopes are the fluorescence microscope, the electron microscope (both, the transmission electron microscope and the scanning electron microscope) and the various types of scanning probe microscopes.

3.2.2 USES AND MAINTENANCE OF THE MICROSCOPE

USES OF THE MICROSCOPE

• Be conversant with the parts of the microscope and their functioning for efficient use.

• Turn on the light source, and adjust the optimum light setting to ensure the correct level of brightness by turning or sliding the brightness adjustment knob at the base.

• Rotate the low power objective into position. Remove the eyepiece, look down the body tube and adjust the mirror and diaphragm setting so that light is reflected up the tube and a circle of evenly illuminated light is visible in the field of view.

• View the specimen with the 10x objective, then with the 40x and then with the 100x oil immersion objective.

Maintenance

1. The usual lab microscope has eye pieces or ocular magnifying x10 and an objectives nose piece carrying x4, x10, and x100 (oil immersion) lenses. Normally the lower power lenses are mounted on the nose pieces, whilst the oil immersion objectives may have mounted or kept separately.

2. Every time it is use, the microscope should be set up to the best optical advantage.

3. Keep in mind the limit to resolution.

3.2.3 STAINING TECHNIQUES

Staining is technique used in microscopy to enhance contrast in the microscopic image. Stains and dyes are frequently used in biological tissues for viewing, often with the aid of different microscopes. Stains may be used to define and examine bulk tissues (highlighting, for example, muscle fibers or connective tissue), cell populations (classifying different blood cells, for instance), or organelles within individual cells. Bacteria have nearly the same refractive index as water, therefore, when they are observed under a microscope they are opaque or nearly invisible to the naked eye. Different types of staining methods are used to make the cells and their internal structures more visible under the light microscope. Microscopes are of little use unless the specimens for viewing are prepared properly. Microorganisms must be fixed & stained to increase visibility, accentuate specific morphological features, and preserve them for future use.

STAINING TECHNIQUES

Direct Staining: The organism is stained and background is left unstained Negative staining: The background is stained and the organism is left unaltered

Stains Are Classified As:

· Simple stain

· Differential stain

· Structural or special stains

Simple Staining: The staining process involves immersing the sample (before or after fixation and mounting) in dye solution, followed by rinsing and observation. Many dyes, however, require the use of a mordant, a chemical compound that reacts with the stain to form an insoluble, coloured precipitate. When excess dye solution is washed away, the mordanted stain remains. Simple staining is one step method using only one dye. Basic dyes are used in direct stain and acidic dye is used in negative stain. A simple staining technique is used to study the morphology better, to show the nature of the cellular contents of the exudates and also to study the intracellular location of the bacteria.

Commonly Used Simple Stains Are:

· Methylene blue

· Dilute carbol fuchsin

· Polychrome methylene blue

DIFFERENTIAL STAINS

Gram Staining Differential Stains use two or more stains and allow the cells to be categorized into various groups or types. Both the techniques allow the observation of cell morphology, or shape, but differential staining usually provides more information about the characteristics of the cell wall (Thickness). Gram staining (or Gram’s Method) is an emprical method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell wall. The Gram stain is almost always the first step in the identification of a bacterial organism, While Gram staining is a valuable diagnostic tool in both clinical and research settings, not all bacteria can be definitively classified by this technique, thus forming Gram variable and Gram indeterminate groups as well.

Gram staining Principles: Gram staining is used to determine gram status to classify bacteria broadly. It is based on the composition of their cell wall. Gram staining uses crystal violet to stain cell walls, iodine as a mordant, and a fuchsin or safranin counterstain to mark all bacteria. Gram status is important in medicine; the presence or absence of a cell wall will change the bacterium’s susceptibility to some antibiotics. Gram-positive bacteria stain dark blue or violet. Their cell wall is typically rich with peptidoglycan and lacks the secondary membrane and lipopolysaccharide layer found in Gram-negative bacteria.

Gram Staining Technique

1. Crystal violet acts as the primary stain. Crystal violet may also be used as a simple stain because it dyes the cell wall of any bacteria.

2. Gram’s iodine acts as a mordant (Helps to fix the primary dye to the cell wall).

3. Decolorizer is used next to remove the primary stain (crystal violet) from Gram Negative bacteria (those with LPS imbedded in their cell walls). Decolorizer is composed of an organic solvent, such as, acetone or ethanol or a combination of both.)

4. Finally, a counter stain (Safranin), is applied to stain those cells (Gram Negative) that have lost the primary stain as a result of decolorization

Gram Reaction Gram:

Positive bacteria are those that are stained dark blue or violet by Gram staining. This is in contrast to Gram-negative bacteria, which cannot retain the crystal violet stain, instead taking up the counter stain (safranin or fuchsine) and appearing red or pink. Gram-positive organisms are able to retain the crystal violet stain because of the high amount of peptidoglycan in the cell wall. Grampositive cell walls typically lack the outer membrane found in Gram-negative bacteria. Gram-negative bacteria are those bacteria that do not retain crystal violet dye in the Gram staining protocol.

3.3 STERILIZATION OF SAMPLES

3.3.1 OVERVIEW OF AN AUTOCLAVE

An autoclave is a pressure chamber used to carry out industrial processes requiring elevated temperature and pressure different from ambient air pressure. Autoclaves are used in medical applications to perform sterilization and in the chemical industry to cure coatings and vulcanize rubber and for hydrothermal synthesis. They are also used in industrial applications, especially regarding composites, see autoclave (industrial).Many autoclaves are used to sterilize equipment and supplies by subjecting them to high pressure saturated steam at 121 °C (249 °F) for around 15–20 minutes depending on the size of the load and the contents. The autoclave was invented by Charles Chamberland in 1879, although a precursor known as the steam digester was created by Denis Papin in 1679. The name comes from Greek auto-, ultimately meaning self, and Latin clavis meaning key, thus a self-locking device.

3.3.2 STERILIZATION OF CULTURE MEDIA

The constituents of culture media, water and containers contribute to the contamination by vegetative cells and spores. The sterilization of media is most commonly achieved by applying heat which is done by looking at the quality and quantity of contamination (i.e the type and load of microorganisms) composition of the media and its pH and size of the suspended particles are the important factors that the success of heat sterilization.this is done at 1210c for 15-20 minutes

3.3.4 STERILIZATION OF WASTE MATERIALS

Used materials in microbiological laboratory are always sterilise to get rid or reduce the microbial load when finally discarded in to the environment, which as result abort pollution and contamination of other useful materials in the environment.

3.4 SERIAL DILUTION

9 ml of distilled water were pipetted into a clean test-tube and were covered with cotton wool and foil and were autoclaved. 1 ml of each dilution was discharged into the centre of the appropriate petri-dish. 10 ml of molten each of the different media used was poured rapidly but carefully mixed by a combination of to-and-fro and circular (clock wise and anti-clockwise).

· It was done at dilution factor of -7 for the nutrient agar and -9 for potato dextrose agar (PDA) to do the bacteria court i then added 1g into the and distilled water prepared earlier on.

· I then inoculate using pour plate by adding 1 ml of the inoculate to the plate then followed by the addition of NA & PDA on the inoculum, after it solidity we then inverted those plates and then put them into incubator.

Original 9 ml H2O 9 ml H2O 9 ml H2O 9 ml H2O

Sample (10–1 dilution) (10–2 dilution) (10–3 dilution) (10–4 dilution)

3.5 BIOCHEMICAL TESTS

3.5.1 CATALASE TEST

This test is used to identify organisms that produce the enzyme, catalase. This enzyme detoxifies hydrogen peroxide by breaking it down into water and oxygen gas. The bubbles resulting from production of oxygen gas clearly indicate a catalase positive result. The sample on the right below is catalase positive. The Staphylococcus spp. and the Micrococcus spp. are catalase positive. The Streptococcus and Enterococcus spp. are catalase negative.

3.5.2 OXIDASE TEST

This test is used to identify microorganisms containing the enzyme cytochrome oxidase (important in the electron transport chain). It is commonly used to distinguish between oxidase negative Enterobacteriaceae and oxidase positive Pseudomadaceae.

Cytochrome oxidase transfers electrons from the electron transport chain to oxygen (the final electron acceptor) and reduces it to water. In the oxidase test, artificial electron donors and acceptors are provided. When the electron donor is oxidized by cytochrome oxidase it turns a dark purple. This is considered a positive result. In the picture below the organism on the right (Pseudomonas aeruginosa) is oxidase positive.

3.5.3 SUGAR FERMENTATION TEST

This test was carried out to determine the ability of the isolates to metabolize sugar with production of acid\gas or gas. The following sugars were prepared and used for the test glucose, maltose, lactose and mannitol. In the test, 0.2g of each of the sugars was dissolved in 20ml of peptone water solution. A pinch of phenol red was added as indicator of the acid production; insert an inverted durham tube in the culture tubes. Incubate at 37oc for 10min.

NOTE :( production of acid is indicated by the change of the colour of the medium to red or pink. If gas is produced it collects in durham’s tube, which rises up in the culture tube.)

3.6.1 SOIL RESPIRATION ANALYSIS

Soil respiration is used to determine the amount of carbondioxide present in the soil. The live microorganisms respire and evolve CO2 in soil which can be measured and assessed as an index of microbial activity in the soil. it is done by weighing 100g of the soil samples into polythene bags using a weighing balance and were put into glass jars, bijou bottles with thread tied to the neck were filled with 10ml of Sodium hydroxide, the bottles were immersed into the weighed soil in the glass jar without touching the soil and was closed tightly. The samples were left for seven days on the work bench for the sodium hydroxide to trap the carbondioxide in the soil samples.

After seven days, the bijou bottles containing the sodium hydroxide were brought out, the sodium hydroxide was poured into two small beakers, 0.5ml of barium chloride was added into each beaker turning the solution milky and a drop of phenolphthalein was added changing the colour to pink, the solution was then titrated against diluted hydrochloric acid and the end point was observed i.e the point at which the pink colour turns colourless. Each end point(titre value) was recorded which would be later computed into an equation used for the calculation of the soil respiration.

Mg CO2/g = Vo-V1 ×1.1

0.09

WHERE, Vo = initial titre value

V1 = final titre value

1.1 = constant

0.09 = Standard dry weight of soil sample

3.6.2 IDENTIFICATION AND ISOLATION OF BACTERIA AND FUNGI FROM SOIL SAMPLES

It is believed that between two and four billion years ago, the first ancient bacteria and microorganisms came about on earth´s oceans. These bacteria could fix nitrogen, in time multiplied, and as a result released oxygen into the atmosphere. This led to more advanced microorganisms, which are important because they affect soil structure and fertility. Soil microorganisms can be classified as bacteria, actinomycetes, fungi, algae and protozoa. Each of these groups has characteristics that define them and their functions in soil.

MATERIALS USED : Soil sample from farmland, nutrient agar (bacteria), potato dextrose agar(fungi), petri dishes, conical flasks, Streptomycin( for PDA), foil paper, ethanol, cotton wool, test tubes, sterile syringes, spirit lamp, distilled water, weighing balance, Incubator, Autoclave.

PROCEDURE : soil sample was collected from the institute farm, 1g of the soil sample was weighed using the weighing balance.

9ml of distilled water was measured into seven clean test tubes using a sterile syringe and were covered with cotton wool.

Nutrient agar and potato dextrose agar were prepared following manufacturer´s specification ( i.e 28g in 1L of distilled water for nutrient agar and 39g in 1L for PDA) into sterile conical flasks and were corked with cotton wool and foil paper. Both the agar and the test tubes were sterilized in an autoclave at 121ºC for 15minutes.

After sterilization, they were allowed to cool, Streptomycin was added into the potato dextrose agar to inhibit the growth of bacteria. Serial dilution was carried out by dissolving the weighed soil sample into the first test tube containing 9ml of distilled water, it was shaken thoroughly and labeled as stock, 1ml from the stock was transferred into the second test tube and mixed thoroughly to make a dilution factor of 101, same procedure was repeated till it get to the final test tube (10-6).

1ml from the last test tube was transferred into clean petri dish in triplicates, same procedure was repeated for another three set of petri dish.10 ml of nutrient agar was poured aseptically into the inoculated petri dishes for the isolation of bacteria while 10ml of the PDA+ Streptomycin was poured aseptically into the second set of the inoculated petri dishes. The mixture were shaken carefully for homogenization. The plates were left for the agar to solidify, they were incubated in an inverted position. The nutrient agar plates were incubated at 37ºC for 24 hours while the PDA plates were incubated at 28ºC for 72hours. After incubation, the plates were observed for the growth of microorganisms, the shapes, sizes, texture, colours were observed and recorded.

The plates were sub cultured by streaking until pure culture was gotten. NA and PDA slants were prepared for the storage of the pure colonies.

From the pure colonies on nutrient agar plates, biochemical tests were carried out such as catalase, oxidase, sugar fermentation test etc. It was observed that Gram negative organisms are the most dominant in the soil samples.

3.6.3 SOIL PH DETERMINATION

To determine the pH of soil, 5g of the dried soil sample was weighed using the weighing balance into a small beaker and was dissolved in 5ml of distilled water, it was stirred for 15 minutes thrice using a stirrer. The pH meter was switched on and calibrated using pH 4and pH 7. The electrode was rinsed with distilled water and wiped with tissue paper, it was inserted into the beaker containing the mixture and the result was read twice from the pH meter and the average was calculated.

3.6.4 SOIL ENZYME ANALYSIS

This involves the determination of enzymes present in the soil in order to know the type of microorganisms that utilizes the enzyme

Four different enzymes are usually tested for; Urease, Phosphatase, Amylase and Dehydrogenase.

For the urease analysis, the following procedures were followed:

1g of fresh soil sample was weighed into 100ml volumetric flask, 1ml of toluene was added to it and allowed to stand for fifteen minutes, 10ml of buffer (pH 7) was then added followed by 5ml of 10% urea solution. The solution was then incubated at 37ºC for 3hours, after incubation, volume was made up to 100ml by the addition of distilled water , it was then mixed and filtered using filter paper. 0.5ml of the filtrate was introduced into 25ml volumetric flask, 5ml of distilled water was added followed by 2ml of phenolate solution and 1.5ml of sodium hypochloride solution. Absorbance of the mixture was read and recorded at 630nm using a spectrophotometer.

Phosphatase analysis

Procedures: 0.1g of air dried soil was measured into 50ml conical flask, 4ml of buffer (pH 6.5), 0.25ml of toluene and 1ml of 0.115M P-nitrophenyl phosphate solution were added. The flask was swirled for few seconds and then incubated at 37ºC for 1hour. After incubation, 1ml of 0.5M calcium chloride and 4ml of 0.5M sodium hydroxide were added to the mixture, the soil suspension was filtered using whatman filter paper. Optical density of the filtrate was measured at 430nm. The standard curve of the P-nitrophenol in water was expressed as mole of P-nitrophenol released per gram dry soil per hour.

Amylase analysis

Procedures: 5g of air dried soil was weighed into 100ml conical flask, 1.5ml of toluene was added, the mixture was shaken and allowed to rest for fifteen minutes at room temperature. 10ml of distilled water was added followed by 5ml of 2% solution of soluble starch, the flask was corked and incubated at 37ºC for 5hours. After 5hours, 10ml of the suspension was centrifuged at 3000rpm for 20minutes, the suspension was then read at 660nm. The chemicals used were weighed and mixed and then used as blank.

Dehydrogenase analysis

Procedures: 1g of air dried soil was weighed into sterile test tube, 0.1g of calcium carbonate was mixed with it. 1ml of 1% Triphiyl tetrazolium chloride (TTC) was added and was shaken. The tube was closed with rubber stopper and incubated at 30ºC for 24 hours. It was filtered with filter paper and extracted with successive aliquots of concentrated methanol. Volume of the filtrate was made up to 50ml by methanol. The optical density of the filtrate was read at 485nm using methanol extract as blank.

3.6.5 ANTIMICROBIAL SENSITIVITY

ANTIMICROBIAL POTENCY OF SOME PLANT EXTRACTS AGAINST Salmonella typhi AND Klebsiella pneumonia

There is a large number of antimicrobial agents available for treating diseases caused by microorganisms. The antimicrobial agents used in medical practice are aimed at eliminating the infecting microorganisms or at preventing the establishment of an infection. To be of therapeutic use, an antimicrobial agent must exhibit selective toxicity ; it must exhibit greater toxicity to the infecting pathogens than to the host organism. Some plant extracts also serves as an antimicrobial agent used in treating diseases, these plants are known as medicinal plants. Their extracts contains some constituents that can inhibit or kill microorganisms such as sapronin, tannin etc.

There are several different procedures used to determine the sensitivity of microorganisms to plant extracts: The Kirby-Bauer disc method and Agar well diffusion method. This antimicrobial sensitivity test is used to determine the minimum inhibitory concentration(the lowest concentration of the plant extract that inhibit the growth of the test organism).

Materials: plant extracts, conical flasks, filter paper, funnel, oven, mullerhinton agar, acetone, 95% ethanol, sterile syringes, cork borer, inoculating pipette, weighing balance, incubator, autoclave.

Procedures : Plant leaves were dried and grinded into powdered form i.e. Hibiscus sabdariffaand Calpurnia aurea(cape plant).

10g, 20g,40g of each of the powdered leaves were weighed respectively using the weighing balance into different sterile conical flasks and were soaked in 100ml of ethanol which serves as the extractant for 48hours.

After soaking, the solution was filtered using a combination of funnel and filter paper, the supernatant was oven dried while the residues were discarded. Muller hinton agar was prepared following the manufacturer specification i.e. in 1L of distilled water. It was sterilized in an autoclave at 121ºC for 15minutes, after sterilization, it was allowed to cool and then poured on sterile petri dishes. Four holes were bored on the solidified agar plates using a sterile cork borer with diameter 8mm. The surface of the solidified agar was swabbed with Salmonellatyphi for hibiscus and cape plant while other plates were swabbed with Klebsiellapneumoniae using a swab stick.

Little volume from each measured extract i.e. from 10g, 20g and 40g was put into the bored holes using a sterile syringe and was labeled appropriately. Water is put into the fourth hole to serve as control. The plates were incubated at 30ºC for 24hours. After incubation, the plates were observed for the zones of inhibition i.e. a clear zone around the holes showing that the extract has been able to inhibit the growth of the test organism.

CHAPTER THREE

4.0 PROBLEMS ENCOUNTERED DURING SIWES

Some of the difficulties I encountered during my industrial training were as follows;

1. Difficulty getting accepted as a trainee: The factory declined severally to accept my industrial training letter claiming they have a lot of IT students allow costing me over 2 week of idleness.

2. Restrictions to carrying out some tests: As an industrial trainee, I was not given access to carry out some tests in the physicochemical restricted laboratory.

3. Understanding the different attitudes of co – workers or the instructor. Finding myself in an entirely new and work environment, understanding and coexisting with colleagues or trainer was a bit challenging.

4. Lack of financial incentives/motivation: There was never a time the institute or Unit Head during my training motivated the student trainees financially despite daily attendance and punctuality.

4.1POSSIBLE SOLUTIONS

1. The company should have an internal library containing the necessary materials. I believe, present age of technology calls for adequate research information and knowledge. This will create room for improvement and effective learning.

2. Intensive and comprehensive class should be organized time to time by the senior workers as this will go a long way in equipping students with adequate knowledge and good understanding of the industrial process and laboratory activities.

3. Incentives such as transport allowance and feeding allowance (where necessary) should be made available by the company to motivate students.

CHAPTER FOUR

5.0 GENERAL APPARAISAL OF THE PROGRAM

I wish to express my profound gratitude to the entire Siwes body that deemed it fit for students to experience the practical aspect of what they are taught within the four walls of their classrooms which will give them more insight and deeper understanding about what they are/will be taught. My SIWES program at IAR&T has really helped me bridge the gap between my theoretical and practical mode of learning.

5.1 WAYS OF IMPROVING THE PROGRAM

Supervisors assigned to each organization should at least visit twice before the program elapse.

The fund provided by ITF to students should be paid if possible during the program as some of the students go financially bankrupt which makes the program stressful and unconducive or even if it should be paid after the program it should be done on time to help them in one way or the other.

5.2 ADVICE FOR THE FUTURE PARTICIPANTS

Students on SIWES should try by all means and avail themselves to their places of work no matter what regardless of them been paid their or not as their aim is to acquire knowledge.

Students on SIWES should adhere strictly to rules and regulations designed by the organization most especially those in companies should adhere strictly to safety regulations.

Students should be good ambassadors of their institutions of learning.

5.3 ADVICE FOR THE SIWES MANAGERS

SIWES management staff should ensure that SIWES supervisors visit students in their places of attachment at least twice.

Funds allocated to students for the program should be released to them as soon as the SIWES supervisors pay their first visit or immediately after the program.

If possible SIWES body should liaise with the organization on the welfare of students on attachment such as accommodations.

5.4 CONCLUSIONS

My entire period of my SIWES program at the IAR&T Ibadan without mincing words was exciting and exhaustive. My experience during this period has broadened my scope of learning hence bridging the gap between my theoretical and practical aspect of learning which was achieved through interaction with staffs of the organization and also other students who are also there on attachment from other schools.

5.5 REFERENCES

Microbiology 5th edition Lansing Prescott.

‘Laboratory manual for developing countries’ by Maurice John G. Holt;Noel R. Krieg; Peter H.A;James T. Staley;Stanley T.Williams (1994). Bergey’s Manual of determinative bacteriology(9th ed.).Lippincott Williams and wilkins.p.11. ISBN 0-683-00603-7

Brock TD (1999).Robert Koch:a life in medicine and bacteriology.Washington DC:American society of microbiology press ISBN 1-55581-143-4

Vickery K,Hu H, Jacombs AS, Bradshaw DA,Deva AK (2013).

Tan, K.Z et al. / American Journal of Agricultural and Biological Sciences 9(3): 342-360, 2014

A Laboratory Manual prepared by Mr Oladapo IAR&T

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