COVENANT UNIVERSITY

ALPHA SEMESTER TUTORIAL KIT (VOL. 2)

P R O G R A M M E : M I C R O B I O LO GY

300 LEVEL

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DISCLAIMER

The contents of this document are intended for practice and learning purposes at the undergraduate

level. The materials are from different sources including the internet and the contributors do not

in any way claim authorship or ownership of them. The materials are also not to be used for any

commercial purpose.

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LIST OF COURSES

MCB311: Microbial Physiology

MCB312: Food Microbiology

MCB313: Clinical Microbiology

MCB314: Soil and Plant Microbiology

MCB315: Analytical Microbiology and Quality Control

MCB316: Immunology

*Not included

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COVENANT UNIVERSITY CANAANLAND, KM 10 IDIROKO ROAD

P.M.B 1023 OTA, OGUN STATE, NIGERIA

TITLE OF EXAMINATION: B.Sc DEGREE EXAMINATIONS

COLLEGE: SCIENCE AND TECHNOLOGY

SCHOOL: NATURAL AND APPLIED SCIENCES

DEPARTMENT: BIOLOGICAL SCIENCES

SESSION: 2014/2015 SEMESTER: ALPHA

PROGRAMME: MICROBIOLOGY COURSE CODE: MCB 311

COURSE TITLE: MICROBIAL PHYSIOLOGY

CREDIT UNIT: 3 TIME: 3 HOURS

INSTRUCTION: Answer any four (4) questions

___________________________________________________________________________

1. a). Differentiate between competitive, uncompetitive and non-competitive inhibitions

(6Marks)

b). Given the formula ∆Go'

= -2.303RT · logKeq, use the relationship between ∆Go'

and Keq to differentiate between endergonic and exergonic reactions (8Marks)

c). Discuss the three (3) types of work that living cells require energy to carry out

(6Marks)

2. a). In a tabular form, list the classes of enzymes, their modes of action and give three

(3) examples each (10Marks)

b). Define the term metabolism. Give the reason why anabolism requires a source of

electrons stored in the form of reducing power (3Marks)

c). List the series of steps involved in anabolism (5Marks)

d). Differentiate between constitutive and inducible enzymes (2Marks)

3. a). Discuss the factors affecting enzyme activity (9Marks)

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b). Explain the models of enzyme-substrate interaction (6Marks)

c). In a simple equation, combine the two laws of thermodynamics, defining the

parameters used (5Marks)

4. a). Discuss the sequence of reactions in the glycolytic pathway (10Marks)

b). Write short notes on the different types of transport of solutes across membranes

(10 Marks)

5. a). Discuss the important features of two major types of bacteria based on their

mode of nutrition (10 Marks)

b). Give three examples of the roles of symbiotic bacteria (10 Marks)

6. a). Discuss the regulation of biochemical pathways (10 Marks)

b). Discuss the different functions of intermediary metabolism in relation to the starting

materials and the state of different biological processes (10 Marks)

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COVENANT UNIVERSITY

CANAANLAND, KM 10 IDIROKO ROAD

P.M.B 1023 OTA, OGUN STATE, NIGERIA

COLLEGE: SCIENCE & TECHNOLOGY

PROGRAMME: MICROBIOLOGY

COURSE TITLE: FOOD MICROBIOLOGY

COURSE UNIT: 3

MCB312 Tutorial Questions

1. Why is Gram positive bacteria more resistant to CO2 than Gram negative bacteria?

2. What is Controlled Atmosphere Packaging? What is its main function?

3. At what water activity value is food considered as non-hazardous?

4. What are the basic nutrients required for microbial growth?

5. List one antimicrobial constituent each present in the following and their mode of action:

• Garlic

• Egg

• Milk

6. In the fermentation of oil bean seed, members of Enterobacteriaceae are usually present at

the beginning of fermentation but disappear at the latter stage. Explain why this happens.

7. List four microorganisms involved in the fermentation of oil bean seed.

8. What are Prebiotics? List three types of Prebiotics.

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9. Give two beneficial uses of prebiotics to man.

10. Enumerate three types of spoilage in canned foods

11. What are the external sources responsible for the microbial contamination in food?

12. Why is it important to identify the sources of microorganisms in foods?

13. In the fermentation of cassava to produce garri, how is the poisonous compound, Hydrogen

cyanide released?

14. Mention 5 specific attributes that position an organism to be an effective probiotic.

15. As a Food Microbiologist working in National Agency For Food and Drug Administration

and Control (NAFDAC), what strategies would you put in place to enforce Food Quality?

16. Why is the Hazard Analysis Critical Control Point (HACCP) system necessary in a Food

Industry?

17. What measures would you employ in the control of food-borne pathogens?

18. Mention five types of bacterial food-borne pathogens and the diseases they cause.

19. What are probiotics?

20. Give 6 examples of Probiotic microorganisms.

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ANSWERS

1. Gram-positive bacteria possess a thick peptidoglycan layer than the Gram negative

bacteria.

2. Controlled Atmosphere Packaging is the storage of food in atmosphere containing 10%

Carbon dioxide. Its main function is to preserve the food thereby extending the shelf-life

of the food.

3. Food is considered to be non-hazardous at a water activity of 0.85

4. The basic nutrients required for microbial growth are: Carbon, Nitrogen, Oxygen,

Hydrogen, Vitamins, minerals and a host of growth factors.

5. Garlic – Allicin

Egg - Ovatransferrin/Avidin/ Lysozyme/Ovoflavoprotein

Milk – Lactaferrin, Lysozyme, Lactoperoxidase

6. Members of the Enterobacteriaceae are usually present at the beginning of the fermentation

where the pH is low but disappear at the later stage because of the increase in pH thereby

leading to succession by other microorganisms suited for an alkaline environment. The

increase in pH is due to proteolysis which leads to the release of amines and amino acids

from the leguminous seed.

7. Microorganisms involved in the fermentation of oil bean seeds include

• Alcaligenesviscolatis

• Pseudomonas aeruginosa

• Micrococcus varians

• Bacillus cereus

8. Prebiotics are non-digestible food ingredients that stimulate the growth and/or activity of

bacteria in the digestive system for the benefit of man’s health.

Examples of Prebiotics incl

Inulin

Fructooligosaccharides (FOS)

Galactooligosaccharides (GOS)

9. Prebiotics increase fecal bifidobacteria

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They increase magnesium and calcium absorption.

10. Three types of spoilage in canned foods include:

• Flat Sour spoilage

• Thermophilic Anaerobe(TA) spoilage

• Sulfide Stinker spoilage

11. Air, soil, sewage, water ,humans, food ingredients, equipment, package and insects

12. The sources of microorganisms in food should be identified in order to:

develop methods to control access of some microorganisms in the food

develop processing methods to kill them in foods.

determine the microbiological quality of foods and in turn set up microbiological

standards and specifications of foods and food ingredients.

13. During fermentation, endogenous linamarase present in cassava roots hydrolyze linamarin

and lotaustralin releasing hydrogen cyanide (HCN).

14. Specific attributes that position an organism to be an effective probiotic include:

acid tolerance

bile tolerance

cell surface hydrophobicity

protoplast regeneration

antimicrobial activity

15. As a Food Microbiologist working with NAFDAC, the following strategies will be

enforced to ensure food quality:

Education & Training

Inspection of facilities & operations

Microbiological testing

Hazard Analysis Critical Control Point(HACCP) System

16. The HACCP System is necessary because:

It is a simple and effective way to ensure food safety.

It allows you to predict risks to food safety and prevent them before they happen.

It is a protective concept

It assures food safety from harvest to consumption.

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It takes into consideration:

• Factors that contribute to most outbreaks

• Risks assessment techniques to identify and prioritize hazards.

17. Measures that must be employed in the control of food-borne pathogens:

Food hygiene must be practiced both by food vendors andconsumerse.g proper

washing/disinfection of hands before preparing food and washing/disinfection of hands

after toilet use.

Raw foods must be separated from ready-to-eat foods especially during storage to prevent

cross-contamination and also separate utensils (knives and chopping boards) must be used

when cooking.

Food must be cooked thoroughly to the correct temperature e.g minced meat products such

as hamburgers should reach a temperature of 700C for at least 2 minutes, eggs should be

boiled until the yolks are firm.

Food must be kept at safe temperature e.g chilled, ready to eat foods must be kept at

temperatures below 5ºC (41ºF), hot foods must be kept at temperatures above 60ºC (140ºF)

before serving.

Safe drinking watermust be used to wash and prepare food and make ice and safe raw

materials must be used for the preparation of food

18. Salmonella typhi – Salmonellosis

Clostridium botulinum- Botulism

Listeria monocytogens- Listeriosis

Vibrio cholerae- Cholera

Campylobacter jejuni- Campylobacteriosis

19. Probiotics are live microorganisms which when administered in adequate amounts confer

a health benefit on the host. They are live nonpathogenic preparation administered to

improve and restore the microbial balance of gastrointestinal tract.

20. Probiotic organisms include:

• Lactic acid bacteria –Lactobacillus spp.

• Bifidobacterium

• A nonpathogenic E. coli strain (E. coli Nissle 1917)

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• Saccharomyces boulardii

• Clostridium butyricum

• Streptococcus salivariussubspecies thermophilus

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COVENANT UNIVERSITY

CANAANLAND, KM 10, IDIROKO ROAD

P.M.B 1023, OTA, OGUN STATE, NIGERIA. TITLE OF EXAMINATION: B.Sc EXAMINATION

COLLEGE: SCIENCE & TECHNOLOGY

SCHOOL: NATURAL & APPLIED SCIENCES

DEPARTMENT: BIOLOGICAL SCIENCES

SESSION: 2014/2015 SEMESTER: ALPHA

COURSE CODE: MCB 313 CREDIT UNIT: 3

COURSE TITLE: CLINICAL MICROBIOLOGY

INSTRUCTION: Answer four questions in all; Answer at least one question from each section

TIME: 2 ½ HOURS

SECTION A

1. a) List the general rules for the collection and transportation of clinical specimens

(10 marks)

b) What are the criteria for rejection of specimens? (4 marks)

c) Describe the precautions to be taken for collection and transportation of the following

samples:

i. Cerebrospinal fluid

ii. Stool (5 marks)

d) Why is modified Cary Blair medium used as a transport medium? (1 mark)

2. a) List five organisms that can be isolated/detected from

i) Blood samples ii) cerebrospinal fluid (CSF) (5 marks)

b) Describe the appearance of fecal specimens in five named bacterial diseases and give their

causative agents. (7.5 marks)

c) Name three culture media for isolating pathogenic bacteria from stool and describe the

appearance of the bacteria on the media. (3 marks)

d) What are the roles of the clinical microbiologist in investigating infectious diseases?

(4.5 marks)

SECTION B

3. (a) List the uses of the following media in a typical clinical bacteriology laboratory:

i. Tellurite blood agar

ii. Dorest egg medium

iii. Cooked medium

iv. Lactose egg yolk medium

v. Deoxycholate lactose agar

vi. Tinsdale medium

vii. Phenylethyl alcohol agar

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viii. Tryptic soy agar

ix. Chocolate agar

x. Blood agar

xi. KIA medium

xii. MacConkey agar

xiii. Mannitol salt agar (13 marks)

(b) Write a note on quality control measures in a typical microbiology laboratory (7 marks)

4. Explain

(a) the following staining techniques

(i) Albert (ii) Giemsa (iii) Ziehl- Neelson (iv) Loeffler’s methylene blue (v) Gram

(7.5 marks)

(b ) the procedures for isolation and identification of following bacteria from clinical

specimens

(i) Streptococcus pyogenes

(ii) Clostridium perfringens (iii) Haemophilus influenzae (iv) Bacillus anthracis (v) Klebsiella pneumonia (7.5 marks)

(c ) procedures for collection of sputum and pus samples (5 marks)

SECTION C

5. a) List the specimens used in the diagnosis of genital infections in a clinical laboratory?

(2 marks)

b) What are the symptoms of urethritis? ii) Mention the types of urethritis and the causative

organisms. (4 marks)

c) Certain sexually transmitted infections are asymptomatic in women, mention them.

(2 marks)

d) A woman reports to the hospital with a vaginal infection, using physical examination of the

vaginal discharge, mention the organisms that may be involved. (ii) What simple tests can be

used to identify the causative organism? (9 marks)

e) List the organisms implicated in genital ulcers. (3 marks)

6. a) What are the complications of gonorrhoea in men and women? (6 marks)

b) Mention the enriched selective medium used in culturing specimens from gonorrhoea, why

is it used? (2 marks)

c) Describe the simplest test used to diagnose syphilis? (3 marks)

d) Differentiate between infections caused by herpes simplex viruses 1 and 2. (ii) What are the

likely manifestations of HSV-2 in an infected newborn? (7 marks)

e) Why is antibiotic susceptibility testing necessary? (2 marks)

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COVENANT UNIVERSITY, OTA- OGUN STATE.

DEPARTMENT OF BIOLOGICAL SCIENCES

MICROBIOLOGY UNIT

MCB 314: SOIL AND PLANT MICROBIOLOGY

ALPHA SEMESTER TUTORIAL QUESTIONS.

QUESTIONS

1.What are “plant diseases” and list the potential causes of plant diseases.

2. Discuss the classification of plant diseases based on the responsible pathogens.

3. Discuss on the term “Epiphytotics” and explain how environmental factors influence

epiphytotics.

4.Discuss any three types of disease symptom that you know.

5.Explain the three basic methods involved in

i) Biological Pest Control

ii)Chemical Pest Control

6. List the various methods by which plant diseases are spread.

7.Define pathogenesis, list the steps involved in pathogenesis and succinctly state Koch’s

Postulates.

8. Define Soil

9. Mention the two types of weathering and list the factors that aid each type.

10.List the factors that aid transformation of mineral matter into soil

11.In a tabular form, write three functions each of the three (3) major elements and symptoms of

their deficiencies in plants.

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12.List the macro and micro- elements and state four (4) differences between them.

13.Explain four (4) factors that affect the rates of nitrification.

14.State three (3) organisms involved in nitrogen fixation.

15.With the aid of a well labelled diagram explain the processes involved in nodulation.

16.State five (5) sources of plant nutrient in the soil.

17.Explain five (5) ways by which nutrients are lost from the soil.

18.Differentiate between endomycorrhiza and ectomycorrhiza giving three (3) examples of

endomycorrhiza.

19.State the types of biofertilizers you know and give three advantages of biofertilizers.

20.What makes orchid mycorrhiza different from the others? Give four (4) ways by which

biofertilizers act.

ANSWERS

1.Plant Diseases are any harmful deviation or alteration from the normal functioning of

physiological processes. It is also defined by some as: Disease is a malfunctioning process that is

caused by continuous irritation which results in suffering.

B) The potential causes of plant diseases are:

i) Animate or biotic causes

ii) Mesobiotic causes iii ) Inanimate or abiotic causes

2.The classification of plant diseases on basis of type of pathogens causing them:

(i)Non-infectious disease or non-parasitic or physiological diseases: These are diseases caused by

natural agencies such as the inanimate or abiotic factors. And they can incite such diseases in plants

under a set of suitable environmental conditions.

The possible causes include:

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a) Weather

b) Nutrient deficiency

c) Toxic substances

a) The Weather: This includes Lightning, Rain/hail stone, Wind, Drought, Flooding, Strong sun,

Frost

b) Nutrient Deficiencies

Deficiency of nutrients can cause symptoms like yellowing, reddening, spotting, stunting, distorted

growth and death. However, symptoms vary with the element involved.

c) Toxic substances in the soil or air:

Too much elements required by plants can cause death, with similar symptoms to deficiencies.

(ii) Infectious disease: Infectious means that which tends to spread from one plant to another or

from one part of the plant to the other and are caused by pathogens.

a. Disease caused by parasitic organisms: The organisms included in animate or biotic causes

can incite diseases in plants.

b. Diseases caused by viruses and viroids.

a) Fungi

Fungi are plants without chlorophyll and must live on other plants which make their own food.

Fungi are generally microscopic but there are large ones like mushrooms. Most fungal bodies

consists of thin delicate filaments or very small tubular structures called hyphae, which grow in or

on the host’s tissue. Sometimes these threads collect together to give big structures like mushrooms

and toad stools. They produce spores which are often visible on the affected parts of the plants.

E.g. rust spores on beans, smut on wheat or maize, and mildews on cucurbits. The fungi that are

known as parasites and saprophytes. The spores serve to spread the fungus to new plants. Examples

of diseases caused by fungi are, Late Blight of Potato, Wheat rust and smut, apple scab etc.

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b) Bacteria

Many destructive plant diseases are caused by bacteria, e.g. wilts, soft rots of fruit and vegetables,

many cancer-like growths of galls of fruits and stems and several leaf spots, e.g. halo blight of

beans. Bacteria are small single- celled structures, that aggregate to form, often characteristic,

cream or yellow slime. The rod-shaped bacteria are the major causes of plant disease. Bacteria

cannot enter directly through the epidermis, as can fungi, but go through wounds or stomata.

c) Viruses

These are the smallest of all and invisible under an ordinary microscope. They are recognizable

only through symptoms they produce together with the fact that such symptoms can be transmitted

to a healthy plant by various means. Virus are not living organisms, but are related to the basic

protein-like components of living matter - the nucleic acids.

d) Nematodes

Nematodes are small worms which can just be seen with the naked eye. Most of them live in the

soil without doing any damage but some are parasitic. Most of the parasitic ones damage roots by

burrowing into them and causing galls or just feeding on them and sucking the contents out of the

root cells.

e) Algae

Algae are single-celled or thread-like plants which contain chlorophyll. Some algae in the tropical

areas are plant parasitic while in general they are not likely to cause any serious damage except

under special circumstances or in neglected plantations of tree crops.

f) Parasitic Plants

Some plants like the mistletoe grow on other plants and derive nutrition thereby causing the host

plant to die.

3.Discussion of Epiphytotics

Is defined as the sudden or abnormally destructive outbreak of a plant disease OR the destruction

of a large number of plants in an area at the same time. In contrast, endemic (ephytotic) diseases

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occur at relatively constant levels in the same area each year and generally cause little concern.

Epiphytotics affect a high percentage of the host plant population, sometimes across a wide area.

They may be mild or destructive and local or regional in occurrence. They result from various

combinations of factors, including the right combination of climatic conditions, when a pathogen

is introduced into an area in which it had not previously existed, when new plant varieties are

produced by plant breeders without regard for all enphytotic diseases that occur in the same area

to some extent each year, some of these varieties may prove very susceptible to previously

unimportant pathogens, when host plants are cultivated in large acreages where previously little or

no land was devoted to that crop. Epiphytotics may occur in cycles such as when a plant disease

first appears in a new area and grows rapidly to epiphytotic proportions. In time, the disease wanes,

and, unless the host species has been completely wiped out, the disease subsides to a low level of

incidence and becomes enphytotic. This balance may change dramatically by conditions that

favour a renewed epiphytotic.

B) Environmental factors affecting disease development:

Important environmental factors that may affect development of plant diseases and determine

whether they become epiphytotic include:

i) Temperature: A pathogen has an optimum temperature for growth. In addition, different growth

stages of the fungus, such as the production of spores, their germination, and the growth of the

mycelium, may have slightly different optimum temperatures. The optimum temperature, usually

combined with optimum moisture condition, permits forecasting, with a high degree of accuracy,

the development of such diseases as blue mold of tobacco, downy mildews of vine crop, lima

beans, amongst others. Effects of temperature may mask symptoms of certain viral and

mycoplasmal diseases, however, making them more difficult to detect.

ii) Relative humidity: Relative humidity is an important factor in the development of some storage

rots e.g. Rhizopus soft rot of sweet potato (Rhizopus stolonifer) is an example of a storage disease

that does not develop if relative humidity is maintained at 85 to 90 percent. High humidity favours

development of the great majority of leaf and fruit diseases caused by fungi and bacteria. Moisture

is generally needed for fungal spore germination, the multiplication and penetration of bacteria,

and the initiation of infection.

iii) Soil moisture: High or low soil moisture may be a limiting factor in the development of certain

root rot diseases. High soil-moisture levels favour development of destructive water mold fungi,

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such as species of Aphanomyces, Pythium, and Phytophthora. Overwatering, by decreasing oxygen

and raising carbon dioxide levels in the soil, makes roots more susceptible to root-rotting

organisms. Diseases such as charcoal rot of corn, sorghum, soybean, e.t.c are most severe under

low soil-moisture levels.

iv) Soil pH: Soil pH, a measure of acidity or alkalinity, markedly influences a few diseases, such

as common scab of potato and clubroot of crucifers. Certain pathogens are favoured by loam soils

and others by clay soils. Phymatotrichum root rot attacks cotton and some other plants; this fungus

is serious only in black alkaline soils, pH 7.3 or above that are low in organic matter.

v) Soil fertility: The raising or lowering of the levels of certain nutrient elements required by plants

frequently influences the development of some infectious diseases example, fire blight of apple

and pear, stalk rots of corn and sorghum. These conditions can be counteracted by adding adequate

amounts of nutrients to reduce acidity or alkalinity such as adding of potash to reduce acidity.

4.Discussion on any three different types of disease symptoms:

i)Necrosis: The term necrosis is used to indicate the condition in which the death of cells, tissues

and organs has occurred as a result of infection by pathogen.

ii)Wilt: This is occurs when the leaves and other green or succulent parts lose their turgidity,

become flaccid and droop. Later the young growing tip or the whole plant may suddenly or

gradually dry up. Wilting may be the result of an injury to the root system, to the partial plugging

of water conducting vessels or to toxic substances secreted by the pathogen and carried to delicate

cells with water. Withering and drooping of a plant which usually starts first from some leaves to

growing tip occurs suddenly or gradually

iii)Mildew: Mildews are plant diseases in which the pathogen is seen as a growth on the surface

of the host. They appear as white, grey, brownish, or purplish patches of varying size on leaves,

herbaceous stems, or fruits. In downy mildews the superficial growth is a tangled cottony or downy

layer, while in the powdery mildews enormous numbers of spores are formed on superficial growth

of the fungus giving a dusty or powdery appearance.

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iv)Rusts: These are diseases with rusty symptoms. The rusts appear as relatively numerous small

pustules of spores, usually growing out through the host epidermis. The pustules may be either

dusty or compact, and red, brown, yellow, or black in colour.

v)Smuts: The affected parts of the plant show a black or purplish-black dusty mass. These

symptoms usually appear on floral organs, particularly the ovary but they can also be found on

stems, leaves and roots.

vi)Scab: The term scab refers to roughened or crust-like lesions or to a freckled appearance of the

diseased organ. In some diseases of this type the parasite appears at a certain stage, in others it is

never seen.

vii)Blotch: This symptom consists of a discolouration giving the leaf, fruit, and other parts a

blotched appearance as in sooty blotch and fly speck disease of apple fruits.

viii)White blisters: Numerous white coloured blister-like ruptures are surfaced on the host

epidermis that forms powdery masses of spores of fungi. They are called white blisters or white

rust.

ix)Colour change: It denotes conversion of green pigment of leaves into other colours mostly to

yellow colour, in patches or covering the entire leaves. (i) Etioliation: Yellowing due to lack of

light, (ii) Chlorosis: Development of yellow colour as a result of low temperature, nutrient

deficiency, excess of lime or alkali, lack of iron, disturbances by fungal and bacterial diseases,

viral infection etc. is known as chlorosis. (iii) Albino: Lack of any pigment and turned into white

or bleached (iv) Chromosis: This is a red, purple or orange pigmentation due to physiological

orders etc.

x)Exudation: This symptom is commonly found in bacterial diseases when masses of bacterial

cells ooze out to the surface of affected plant parts and form some drops or as thin smear, it is

called exudation. This exudation forms a crust on the host surface after drying.

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xi)Overgrowth: This is the abnormal increase in size of plant organs or the entire plant as a result

of stimulation of the host tissues. This may be brought about either or both of the two processes,

hyperplasia and hypertrophy. Hyperplasia is the abnormal increase in the size of a plant organ

due to increase in number of cells of which the organ is composed. In hypertrophy, the increased

size of the organ is due to increase or excessive enlargement of the size of cell of a particular tissue.

xii)Atrophy or dwarfing: This is a disease symptom in which the plants remain stunted or dwarf

because of growth inhibition due to reduction in cell division or cell size. It is possible that

hypertrophy and atrophy both can occur in the same organ.

5) The three basic types/methods involved are:

i) Biological Pest Control: Biological control of disease employs natural enemies of pests or

pathogens to eradicate or control their population. This can involve the introduction of exotic

species, or it can be a matter of harnessing whatever form of biological control exists naturally in

the ecosystem in question. The induction of plant resistance using non-pathogenic or incompatible

micro-organisms is also a form of biological control. There are three basic types of biological pest

control strategies: importation (sometimes called classical biological control), augmentation and

conservation.

Importation

Importation involves the introduction of a pest's natural enemies to a new locale where they do not

occur naturally. This is usually done by government authorities. These introduced pests are

referred to as exotic pests and it involves determining the origin of the introduced pest and then

collecting appropriate natural enemies associated with the pest or closely related species. Selected

natural enemies are then passed through a rigorous assessment, testing and quarantine process, to

ensure that they will work and that no unwanted organisms are introduced.

Augmentation

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It involves the supplemental release of natural enemies, thereby boosting the naturally occurring

population. Relatively few natural enemies may be released at a critical time of the season

(inoculative release) or millions may be released (inundative release). An example of inoculative

release occurs in greenhouse production of several crops. Periodic releases of the parasitoid,

Encarsia formosa, are used to control greenhouse whitefly, and the predatory mite Phytoseiulus

persimilis is used for control of the two-spotted spider mite. Lady beetles, lacewings, or parasitoids

such as those from the genus Trichogramma are frequently released in large numbers (inundative

release).

Conservation

It involves the adaptation of the natural enemies to the habitat and to the target pest, and their

conservation can be simple and cost-effective. Cropping systems can be modified to favor the

natural enemies, a practice sometimes referred to as habitat manipulation. Providing a suitable

habitat, such as a shelterbelt, hedgerow, or beetle bank where beneficial insects can live and

reproduce, can help ensure the survival of populations of natural enemies.

ii)Chemical Pest Control: Chemicals such as herbicides, insecticides, are very important in plant

diseases. Three groups of chemicals include:

Herbicides

Herbicides are used to control the growth of unwanted plants (weeds) and generally act by

restricting growth. They inhibit the action of one or more of the many receptors that catalyze

reactions which are essential to the growth of the plant. There is one group however, the auxins,

that kill by overstimulating growth. With selective herbicides, either the target in the weed is

affected more than that of the crop, the herbicide is degraded more quickly within the crop, or the

uptake or translocation of the active ingredient differs from that of weeds. Non-selective

herbicides kill crops as well as weeds.

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Insecticides

These include compounds interfering with the nervous system of pest invertebrates, since this

target organ usually provides rapid control. Insecticides acting on target sites such as

acetylcholinesterase (organophosphates and methyl carbamates), voltage-gated sodium channels

(pyrethroids), nicotinic acetylcholine receptors (neonicotinoids) and ligand-gated chloride

channels (macrocyclic lactones and phenylpyrazoles) account for more than 75% of total

insecticides sales.

Fungicides

They are synthetic chemical compounds that kill fungi or inhibit their growth. Some biological

organisms can also be used to control fungal infections, which include mildews, rusts and leaf

spots. Some fungicides offer protection against the development of fungi, and others cure the plant

by eliminating the fungus. Nearly all modern fungicides (except for inorganic salts) degrade in

aerobic soils and therefore they are environmentally friendly, the rate of degradation depending on

the temperature and humidity.

6) The various means by which plant diseases are spread are:

i)Spread through seeds and other planting materials such as tubers, rhizomes, e.t.c.

ii)Spread by natural agents such as wind, water, rain.

iii)Spread by animals and insects.

iv)Spread by people.

7.Pathogenesis is defined as the entire chain of events leading to the development of a disease.

The steps involved in pathogenesis are:

i) The pathogen perennates at some location during the absence of cultivated host.

ii) It gets transported to the cultivated host through some agencies.

iii) The pathogen breaks the host barrier to establish infection.

iv) Effects the host physiology, damages the plant and the plant expresses symptoms.

v) The pathogen finds an exit from the host through its propagules.

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Koch’s Postulate are:

1.Recognition: The pathogens must be found associated with the disease in the diseased plant and

the symptoms recorded.

2. Isolation: The pathogen should be isolated grown in pure culture in artificial media as well as

recording the cultural characteristics.

3.Inoculation: The pathogen of pure culture must be inoculated on healthy plant of some species.

It must be able to reproduce disease symptoms on the inoculated plant identical to the ones earlier

recorded.

4.Re-isolation: The pathogen must be isolated from the inoculated plant in culture media and its

cultural characteristics must be similar to previous isolates.

8. Definition of soil:

Soil can be defined as the solid material on the Earth’s surface that results from the interaction of

weathering and biological activity on the parent material or underlying hard rock.

9.The two types of weathering are:

i)Physical weathering

ii)Chemical weathering

10.The factors aiding the transformation include:

i)Parent material

ii)Climate

iii)Relief

iv)Organisms

v)Man

24

vi)Time

11. Nitrogen (N). Phosphorus (P) and potassium (K) are the three most important nutrients

required for plant growth.

Element

Function Symptoms of deficiency of

deficiency

Nitrogen • Necessary for plant growth

• Necessary for protein production by

plant

• Necessary for critical plant functions

Nitrogen deficiency causes a

yellowing of the leaves and

stunted growth.

Phosphorus • Promotes early root formation and

growth

• Improves the quality of many fruits,

vegetables and grain crops

Stunted growth, reddish or

purple colour on young

plants at low temperatures.

Dead areas may develop on

leaves, fruits and stems

Potassium • Essential for protein synthesis and

cell division.

• Important in fruit formation

• Decreases water requirements of

plants

Poorly developed root

systems and weak stalks.

Seeds and fruits are small and

shrivelled.

12. Differences between macro and micro Elements

Macro elements Micro elements

They occur in easily detectable quantities They occur in very small amounts

They build up plant body and different

protoplasmic constituents

They do not have any such roles

They are not toxic in slight excess Toxic in slight excess

Some accumulate in cell and take part in

different osmotic potential

They are found in traces only and have no

significant role in developing osmotic

potential

Concentration per gram of dry matter is at

least 1mg per 1000microgram

Concentration is less than 1mg/gram of

dry matter

C, H, O, P, K, S, Mg, Fe, Ca. Zn, Mn, B, Cu, Mo and Cl.

13.The rates of nitrification reaction are highly dependent on a number of environmental

factors. These include the substrate and oxygen concentration, temperature, pH, and the

presence of toxic or inhibiting substances.

25

i.Temperature Dependency: Like all microbes, nitrifying bacteria are temperature

sensitive. Rapid changes in temperature do not produce rapid changes in growth rates.

ii. Oxygen Concentration: Nitrifying bacteria are especially sensitive to low oxygen

concentrations, and having enough oxygen is very important.

iii.pH Dependency: pH has strong effect on nitrification rates. The reactions occur fastest when

pH is from 8 – 9

iv.Inhibiting Substances: Many substances can potentially inhibit the nitrification

reactions. Metals are particularly strong inhibitors of the reactions. When exposed to more than

one inhibitor, the extent of inhibition increases greatly.

14.Nitrogen fixation is a process in the nitrogen cycle whereby atmospheric nitrogen is converted

to utilizable forms my micro-organisms. The organisms involved in this include Rhizobium,

Bradyrhizobium, Azospirillum,Azotobacter generally referred to as rhizobia.

15.The processes involved in nodulation include

a. Chemical recognition of roots and Rhizobium

b. Root hair curling

c. Formation of infection thread

d. Invasion of roots by Rhizobia

e. Cortical cell divisions and formation of nodule tissue

f. Bacteria fix nitrogen which is transferred to plant cells in exchange for fixed carbon

26

16.Mineral nutrients can be lost from the soil system and become unavailable for plant uptake.

Nutrient losses occur through:

i.Runoff – loss of dissolved nutrients in water moving across the soil surface

ii.Erosion – loss of nutrients in or attached to soil particles that are removed from fields by wind

or water movement

iii.Leaching – loss of dissolved nutrients in water that moves down through the soil to

groundwater or out of the field through drain lines

iv.Gaseous losses to the atmosphere – primarily losses of different N forms through volatilization

and denitrification

v.Crop removal – plant uptake and removal of nutrients from the field in harvested products

17.Plants obtain mineral nutrients through root uptake from the soil solution. Sources of these

soluble nutrients in soil include:

27

i. Decomposition of plant residues, animal remains, and soil microorganisms

ii. Weathering of soil minerals

iii. Fertilizer applications

iv. Manures, composts, biosolids (sewage sludge), kelp (seaweed), and other organic

amendments such as food processing byproducts

v. N-fixation by legumes

vi. Ground rock products including lime, rock phosphate, and greensand

vii. Inorganic industrial byproducts such as wood ash or coal ash

viii. Atmospheric deposition, such as N and S from acid rain or N-fixation by lightning

discharges

ix. Deposition of nutrient-rich sediment from erosion and flooding

18.The fungus in endomycorrhiza penetrates the cortical cells of the roots of plants while in

ectomycorrhiza, the colonization is characterized by an external coat of fungal hyphae that may

completely cover the host root and the presence of hyphae between root cortical cells that

constitute a structure called "Harting Net". Examples of the endomycorrhiza are

ArbuscularMycorrhiza, Ericoid Mycorrhiza and Orchid Mycorrhiza.

19.The types of biofertilizers

a. Nitrogen fixing biofertilizers e.g. Rhizobium, Bradyrhizobium, Azospirillum and

Azotobacter.

b. Phosphorus solubilising biofertilizers (PSB) e.g. bacillus, Pseudomonas,

AspergillusandEnterobacter.

c. Phosphate mobilising biofertilizer e.g. Mycorrhiza.

d. Plant Growth Promoting Biofertilizers e.g. Pseudomonas spp.

The advantages of biofertilizers are,

a. They are cost effective relative to chemical fertilizers.

b. They are environmentally friendly.

c. They provide better nourishment to the plant.

20.Orchid mycorrhizal fungi form a type of association with their host plant that is very different from all

other types of mycorrhizal associations. Orchid mycorrhizal fungi are essential for the early stages of

growth of the plant when the plant is totally dependent on the fungus for carbon.

How biofertilizers work:

28

a. They fix atmospheric nitrogen in the soil and root nodules of legume crops and make it

available to plants.

b. They solubilise the insoluble forms of phosphates like tricalcium, iron and aluminium

phosphates into available forms.

c. They scavenge phosphates from soil layers.

d. They provide hormones and anti-metabolites which promote root growth.

e. They decompose organic matter and help in mineralisation in the soil.

f. When applied to seed or soil, biofertilizers increase the availability of nutrients and

improve yield without adversely affecting the soil and environment.

29

COVENANT UNIVERSITY CANNANLAND, KM 10 IDIROKO ROAD

P.M.B. 1023 OTA, OGUN STATE, NIGERIA

SESSION: 2014/2015 SEMESTER: Alpha Semester COLLEGE: SCIENCE & TECHNOLOGY DEPARTMENT: BIOLOGICAL SCIENCES PROGRAMME: MICROBIOLOGY COURSE CODE: MCB 315 COURSE TITLE: ANALYTICAL MICROBIOLOGY & QUALITY CONTROL

TUTORIAL REVIEW QUESTIONS WITH SELECTED MODEL ANSWERS

1a.) Quality Assurance incorporates Good Manufacturing Practice (GMP), In-Process Control

and Quality Control.

(i). Define GMP and describe what it means in practice.

GMP is the part of QA which is aimed at ensuring the product is consistently

manufactured to a quality appropriate for its intended use and to meet the requirements of

regulatory authorities

In practice, this means that:

Personnel must be adequately trained.

Suitable premises and equipment must be employed.

Correct materials must be used.

Approved procedures must be adopted.

Suitable storage and transport facilities must be available.

Appropriate records must be made.

(ii) Discuss the major concerns of Quality Control in GMP.

QC is that part of GMP that is concerned with sampling, specifications and testing.

QC also includes the organization, documentation and release procedures which ensure

that the necessary and relevant tests are carried out, and that materials are not released for

use, nor products released for sale or supply, until their quality has been judged

satisfactory. QC must not be confined to laboratory operations only. It must be included

30

in all decisions that may affect the quality of the product. For the satisfactory operation of

QC, it must be independent from production.

b.) Describe the implications and the necessary actions you, as an Analytical Microbiologist,

would take if the following were detected during routine environmental monitoring of

your industrial plant:

(i) Staphylococcus spp.

(ii) Bacillus spores

The presence of Staphylococcus spp. suggests human-borne contamination

The adequacy of the changing facility and gowning would then be checked.

In contrast, Bacillus spores would suggest environmental contamination

In this case, the entry of equipment into the cleanroom would be scrutinized.

2. The Standard Organization of Nigeria (SON) established the Nigerian Standard for Drinking

Water Quality (NSDWQ)

a) What are the objectives of the NSDWQ?

b) Describe the five physical and four microbiological parameters and maximum allowable limits

set in the NSDWQ.

3) Microorganisms contaminating manufacturing plants can cause product spoilage and/or if

pathogenic, they can initiate infections.

a) List five potential sources of microbial contamination in an industrial manufacturing

plant.

Working surfaces, fixtures and equipment

Pooled stagnant water

Fall-out of dust- and droplet-borne organisms

Poor hygiene, communicable diseases, and open lesions on exposed body surfaces

of personnel

Untreated raw materials that are derived from natural sources (animals, plants)

31

Processes employed in product manufacture (e.g. concentration of the level of

spore-forming bacteria through drying)

Packaging used for raw materials, such as unlined paper sacks which may absorb

moisture and be subject to microbial deterioration

Variation in temperature of raw material during storage

(b) Discuss five measures by which microbial contamination of a pharmaceutical product

can be minimized.

Measures by which microbial contamination of a pharmaceutical product can be

minimized include:

Risk Assessment:

Manufacturers are expected to demonstrate extensive assessment of risk to the regulatory

authorities.

This proactive risk analysis should be driven by a sound understanding of the process,

and microbial ecology of the environment and ingredients.

A widely used method in the food industry which is becoming common in the

pharmaceutical industry is Hazard Analysis Critical Control Points (HACCP).

Environmental Cleanliness and Hygiene:

The spread of dust during manufacture or packaging must be avoided.

The manufacture of certain liquid preparations and creams and ointments should be in a

closed system. This serves to prevent microbial contamination and evaporative loss.

Adequate hand-washing and hand-disinfecting facilities and protective garments,

including headgear and gloves, must be provided.

Quality of Starting Materials:

Materials of good microbiological quality must be selected.

Raw materials must be free from Escherichia coli and Salmonella spp.

Precautions must be taken to ensure that dry raw materials are held below the minimum

water activity levels.

Polythene-linked sacks must be used for packaging of raw materials.

32

A sterilization stage must be included either before or during the manufacturing process.

Water:

Water must be treated by filtration, or by chemicals, to render it free from coliforms.

Water that are virtually apyrogenic must be used for parenteral products.

An endotoxin level must not be more than 0.251 IU/ml

Appropriate grade of water must be used in pharmaceutical manufacturing.

Process Design:

The manufacturing process must be fully defined.

The facilities available must support the implementation of the manufacturing process.

The manufacturing process must be capable of providing product that is

microbiologically acceptable, and conforms to specifications.

Quality Control and Documentation:

Operators must be adequately trained.

The aseptic techniques of the operators must be monitored by both observation and

microbiological testing.

Air filter and sterilizer efficiency must be evaluated. Final tests on the finished product

must be conducted sterility testing testing for pyrogens (where necessary).

Packaging, Storage and Transport:

Packaging must serve its proper functions; it should keep the contents in, keep

contaminants or moisture out, be labeled to permit identification of its contents.

4. Discuss the requirements for clean and aseptic areas of a manufacturing plant with

regards to:

a) Internal surfaces, fittings and floors

33

b) Services, equipment and operation

5a.) The National Agency for Food and Drug Administration and Control (NAFDAC) is a

Nigerian Federal Government regulatory Agency. List five mandates that NAFDAC is

authorized to implement on behalf of the government.

In accordance with the enabling laws, NAFDAC is authorised to:

Regulate and control the importation, exportation, manufacture, advertisement, distribution, sale

and use of regulated products.

Conduct appropriate tests and ensure compliance with standard specifications.

Undertake appropriate investigation of the production premises and raw materials of regulated

products.

Compile standard specifications, regulations, and guidelines for the production, importation,

exportation, sale and distribution of regulated products.

Control the exportation and issue quality certification of regulated products intended for export.

Establish and maintain relevant laboratories for the performance of its functions.

Ensure that the use of narcotic drugs and psychotropic substances are limited to medical and

scientific use only.

b.) Recently, a local Daily News Paper ran a story about Company X which is reportedly

manufacturing and selling a drug that was not duly licensed or registered with NAFDAC.

Discuss five actions the Enforcement Directorate of the Agency might take to address the

potential contravention of Act 25 of 1999.

Enforcement Directorate is responsible for the investigation, interrogation and compilation of

case files in the Agency.

Charged with the responsibility for enforcing the provisions of Act. 25 of 1999. Some of this

provisions include:

Prohibition to produce , import , manufacture , distribute, display for the purpose of sale

any counterfeit , adulterated , banned , fake, substandard or expired drug or unwholesome

processed food.

Prohibition of sale or hawking of drugs or poisons in any place not duly licensed or

registered by appropriate authority.Conducting surveillance on companies and persons

suspected to be violating NAFDAC’s regulations.

34

Carrying out investigations on such persons and companies

Interrogation of suspects

Sampling of NAFDAC regulated products to the laboratory.

Compilation of case files.

Raiding of drug hawkers

Destruction of fake and spurious regulated products

Co-ordination of activities of State Task forces on psychotropic substances.

6. Contrast between settle plate method and silt-to-agar sampler method for monitoring air for

microorganisms in an industrial plant.

7. a) Using the sub-headings: type of pharmaceutical products, criteria for microbiological

quality, contrast non-sterile from sterile medicines.

b) Using an illustration, contrast between products that have been terminally sterilized and

those that have not.

Type of pharmaceutical

products

Criteria for microbiological

quality

Non-sterile medicines Drug products given by

mouth or placed on the skin.

May contain some

microorganisms with limits

on type and concentration,

controlled by specifications.

Sterile medicines Injections, ophthalmic

preparations, and products for

other anatomical sites (e.g.

nose, ear, vagina and bladder)

No detectable

microorganisms.

The product batch should

pass a test for sterility.

Injections are also subject to a

test for pyrogens.

35

b) Distinction between products that have been terminally sterilized and those which have not

8. A widely used method in the food industry which is becoming common in the pharmaceutical

industry is Hazard Analysis of Critical Control Points (HACCP). Describe the seven principles

of HACCP and discuss five of their applications.

9. a) List five potential sources of microbial contamination in an industrial manufacturing plant.

b) Discuss five measures by which microbial contamination of a pharmaceutical product can

be minimized.

• Working surfaces, fixtures and equipment

• Pooled stagnant water

• Fall-out of dust- and droplet-borne organisms

• Poor hygiene, communicable diseases, and open lesions on exposed body surfaces of

Untreated raw materials that are derived from natural sources (animals, plants).

• Processes employed in product manufacture (e.g. concentration of the level of spore-

forming bacteria through drying)

• Packaging used for raw materials, such as unlined paper sacks which may absorb

moisture and be subject to microbial deterioration

• Variation in temperature of raw material during storage

36

• personnel

b) Measures by which microbial contamination of a pharmaceutical product can be minimized

include:

Risk Assessment

• Manufacturers are expected to demonstrate extensive assessment of risk to the regulatory

authorities.

• This proactive risk analysis should be driven by a sound understanding of the

– process, and

– microbial ecology of the environment and ingredients.

• A widely used method in the food industry which is becoming common in the

pharmaceutical industry is Hazard Analysis Critical Control Points (HACCP).

Environmental Cleanliness and Hygiene

• The spread of dust during manufacture or packaging must be avoided.

• The manufacture of certain liquid preparations and creams and ointments should be in a

closed system.

– This serves to prevent microbial contamination and evaporative loss.

• Adequate hand-washing and hand-disinfecting facilities and protective garments,

including headgear and gloves, must be provided.

Quality of Starting Materials

• Materials of good microbiological quality must be selected.

• Raw materials must be free from Escherichia coli and Salmonella spp.

• Precautions must be taken to ensure that dry raw materials are held below the minimum

water activity levels.

37

• Polythene-linked sacks must be used for packaging of raw materials.

• A sterilization stage must be included either before or during the manufacturing process.

Water

• Water must be treated by filtration, or by chemicals, to render it free from coliforms.

• Water that are virtually apyrogenic must be used for parenteral products

– An endotoxin level must not be more than 0.251 IU/ml

– Appropriate grade of water must be used in pharmaceutical manufacturing (see

Table on next slide).

Process Design

• The manufacturing process must be fully defined.

• The facilities available must support the implementation of the manufacturing process.

• The manufacturing process must be capable of providing product that

– is microbiologically acceptable, and

– conforms to specifications.

Quality Control and Documentation

• Operators must be adequately trained.

• The aseptic techniques of the operators must be monitored by both observation and

microbiological testing.

• Air filter and sterilizer efficiency must be evaluated.

• Final tests on the finished product must be conducted

38

– sterility testing

– testing for pyrogens (where necessary)

Packaging, Storage and Transport

– Packaging must serve its proper functions; it should keep the contents in, keep

contaminants or moisture out and be labeled to permit identification of its

contents.

10. Sterility tests attempt to achieve, as far as possible, the continuous monitoring of a particular

sterilization process. Yet, no sterility test provides guarantee as to the sterility of a batch.

a) Discuss five reasons that support the limitations of sterility testing.

b) Describe five applications and roles of sterility testing in GMP.

11.What are the types of hazards and list 5 groups of hazardous chemicals

The types of hazards are:

a) Physical

b) Mechanical

c) Radiation

d) Biological

e) Noise

f) Pressure

B) Groups of hazardous chemicals are:

a) Sensitizers

b) Hepatotoxins

c) Nephrotoxins

d) Neurotoxins

e) Agents which act on the hematopoietic (blood production )system

f) Agents which damage the lungs, skin, eyes, or mucous membranes.

39

2. Define chemical hygiene

Definition of chemical hygiene

This refers to conditions and practices that serve to promote or preserve health while working with

hazardous chemicals.

3. List classes of time-sensitive hazardous chemicals

Time-sensitive hazardous chemicals are: Butadiene, Divinylacetylene Tetrafluoroethylene,

ChloropreneIsopropyl etherVinylidine chloride.

4. List 10 examples of reproductive toxins

1. Types of reproductive hazardous chemicals are:

a) Arsenic

b) Benzene

c) Boric acid

d) Chloroform

e) DDT

f) Dibenzofuran

g) Ethylene glycol

h) Zinc sulfate

i) Toluene

j) Vinyl chloride

k) Hydrazine

l) Lead compounds

m) Lithium

n) Methylene chloride

o) Xylene

5. What are hazard control measures?

Hazard control measures are:

a) Administrative

b) Engineering

40

c) Personal Protection

6. What are coliforms and list seven genera of coliforms.

Coliform bacteria are aerobic and facultative anaerobic, gram negative, non-spore forming,

rod-shaped bacteria that produce gas upon fermentation in prescribed culture media within 48 hrs

at 35°C.

Genera of coliforms include: Escherichia coli, Citrobacter, Enterobacter, Klebsellia,

Erwinia, Pantoea, Serratia amongst others.

7. State the criteria for indicator organisms.

) Criteria for Indicator organisms

a) Should be present whenever enteric pathogens are present.

b) Should be useful for all types of water.

c) Should have a reasonable longer survival time than the hardest enteric pathogen.

d) Should not grow in water.

e) Testing method for the organism should be easy to perform.

f) Density of the indicator should have some direct relationship to the degree of faecal

pollution.

g) Should be a member of the intestinal micro-flora of humans and warm-blooded

animals

8. State the deficiencies of coliform bacteria as an indicator organism of water quality

The Deficiencies of Coliforms as indicator organisms are:

a) Regrowth in aquatic environments.

b) Regrowth in distribution systems, including biofilm colonization.

c) Suppression by high background bacterial growth.

d) Not indicative of health threat.

e) No relationship with enteric protozoan and viral concentration.

9. Discuss on the two laboratory procedures for testing water samples for faecal contamination.

The following steps are important in the collection of water samples:

41

a) Preparation of sample containers.

b) Preparations made before leaving for the sampling site.

c) Collection and Storage of sample.

d) Transportation of sample.

e) Analysis of the sample in the laboratory.

10. List the steps involved in water sample collection

a) Multiple tube fermentation technique: The MPN of total coliform bacteria, faecal

coliform

bacteria, faecal coliform bacteria present in the water sample is determined, along with the

presence / absence of Escherichia coli. It requires a series of tubes containing a suitable selective

broth culture medium (lactose-containing broth, such as MacConkey broth) is inoculated with test

portions of a water sample. The results of replicate tubes and dilutions are reported in terms of the

Most Probable Number(MPN) of organisms present. After a specified incubation time at a given

temperature, each tube showing gas formation is regarded as “presumptive positive” since the gas

indicates the possible presence of coliforms. However, gas may also be produced by other

organisms, and so a subsequent confirmatory test is essential. The two tests are known respectively

as the presumptive test and the confirmatory test. For the confirmatory test, a more selective culture

medium (brilliant green bile broth) is inoculated with material taken from the positive tubes. After

an appropriate incubation time, the tubes are examined for gas formation as before. The most

probable number (MPN) of bacteria present can then be estimated from the number of tubes

inoculated and the number of positive tubes obtained in the confirmatory test using specially

devised statistical tables(McCrady’s Table). The next step is the complete step to inoculate onto

solid media such as MacConkey agar to identify the characteristic morphological pattern of both

total coliforms and faecal coliforms.

b) Membrane filtration method for total coliform and thermotolerant (faecal) coliforms:

The method is based on the filtration of a known volume of water through a membrane

42

filter consisting of a cellulose compound with a uniform pore diameter of 0.45 or 0.2 μm; the

bacteria are retained on the surface of the membrane filter. The membrane containing the

bacteria is incubated in a sterile container at an appropriate temperature with a selective differential

culture medium, characteristic colonies of coliforms/ thermotolerant coliforms are counted

directly.

11. Explain the steps involved in the LAL test for detecting endotoxins in food samples.

The Procedure For LAL Test in detecting Endotoxins.

Limulus amebocyte lysate (LAL) is an aqueous extract of blood cells (amoebocytes) from the

horseshoe crab, Limulus polyphemus. LAL reacts with bacterial endotoxin or lipopolysaccharide

(LPS), this reaction is the basis of the LAL test, which is used for the detection and quantification

of bacterial endotoxins. The most commonly used method for detecting the reaction of LAL and

endotoxin is the gel-clot assay whereby equal volumes of sample and LAL (typically 0.1 ml each)

are combined in glass tube. After an incubation period of 60 min at 37°C, the tubes are inverted

180°. A positive result is indicated by a clot that withstands the inversion. By titrating the LAL

with serially diluted standard endotoxin, the minimum endotoxin concentration required to yield a

positive clot can be determined. This minimum endotoxin concentration, or end point, is referred

to as the LAL sensitivity. The gel-clot assay can be used as a purely qualitative limit test to rank

samples as either positive or negative.

The turbidimetric assays measure the increase in turbidity as a function of the endotoxin

concentration. Endotoxin concentrations in unknowns are determined by comparing the resultant

turbidity with a standard curve. Since the occurrence of turbidity precedes gelation, these assays

are more sensitive than the gel-clot assay. Depending on whether the turbidity is measured at

the end of an incubation period or throughout the reaction, the turbidimetric assays can be

performed as an end-point assay or kinetic assay. The sensitivity of the kinetic assay is about 0.01-

0.001 EU/ml.

The chromogenic assays use a synthetic chromogenic peptide as substrate for the clotting enzyme

in place of coagulogen. The chromogenic substrate is hydrolyzed by the clotting enzyme, releasing

43

the terminal chromogenic moiety with a yellow colour. The chromogenic end-point assay is

normally performed in two steps, in which LAL and sample are first incubated, and substrate is

then added. The resultant absorbance is determined spectrophotometrically after the reaction is

stopped with acetic acid. The chromogenic kinetic assay utilizes a single colyophilized

LAL/substrate reagent, which is incubated with the sample and monitored spectrophotometrically

for the appearance of hydrolyzed substrate. Both the end-point and kinetic assays have a sensitivity

of 0.01 EU/ml.

12. Mention any mycotoxins stating the microorganisms producing them in foods and discuss in

detail the extraction process involved in mycotoxin detection.

Mycotoxins with the microorganisms producing them.

Mycotoxins Microorganisms

Aflatoxin B1 Aspergillus flavus

Aflatoxin B2 Aspergillus flavus

Aflatoxin G1 Aspergillus flavus

Aflatoxin G2 Aspergillus flavus

Fumonisin Fusarium moniliforme

Zearalenone Fusarium graminearum

Nivalenol Fusarium graminearum

Ochratoxins Aspergillus ochraceus

44

ii) Extraction Process For Mycotoxins:

This involves the removal of most of the mycotoxin from the food matrix as possible into a solvent

suitable for subsequent cleanup and determination. In all extraction procedures it is assumed that

the mycotoxin will be distributed evenly among the liquid phase and excluded from the solid phase

of the mixture. Foods are typically extracted with mixtures of water and relatively polar solvents

(acetone, acetonitrile, ethyl acetate, and methanol). Acetonitrile and methanol are the most

common solvents used for extracting the major mycotoxins, with the notable exception of patulin

for which ethyl acetate is preferred. There are two widely used approaches for extraction, either

high speed blending with a solvent for a few minutes or shaking with a solvent for 30 minutes to

2 hours. Once the solid sample has been shaken or blended with the extraction solvent, the liquid

is separated from the solids either by filtration or centrifugation. The extract is then either cleaned

up further to isolate the toxins, or applied directly to the determinative step in the procedure.

45

COVENANTUNIVERSITY

CANAANLAND, KM 10, IDIROKO ROAD

P.M.B 1023, OTA, OGUN STATE, NIGERIA TITLE OF EXAMINATION: B. Sc. EXAMINATION

COLLEGE: SCIENCE & TECHNOLOGY

SCHOOL: NATURAL AND APPLIED SCIENCES

DEPARTMENT: BIOLOGICAL SCIENCES

SESSION: 2014/2015 SEMESTER: ALPHA

COURSE CODE: MCB 316 CREDIT UNIT: 3

COURSE TITLE: IMMUNOLOGY

INSTRUCTION: ANSWER FOUR QUESTIONS IN ALL, TWO QUESTIONS FROM EACH

SECTION. TIME: 3 HOURS

SECTION A

FIGURE A FIGURE B

1. Figure A above represents the elements of the immune system.

a. State one important element that is not represented in the diagram and give five

examples of how your answer protects the body from infection.

(4¾ marks)

46

b. State three examples of each components of the innate defence and briefly

describe how one example contributes to maintaining good health.

(6½ marks) c. State two examples of each components of the adaptive defence and briefly

describe how one example contributes to maintaining good health.

(5 marks) d. Outline five functions of antibodies. (3¾

marks)

2. Figure B above represents the activities of the complement system.

a. State the complement activation pathway depicted in the picture. (2

marks) b. Exhaustively label each of the components in the diagram in a sequential order

beginning from the components immediately after the labelled antigen and

antibody. (9 marks)

c. Briefly describe activities resulting in the swelling and bursting of the cell at the

end of the diagram.

(3 marks) d. Describe the major functions of the complement system in the body’s immunity.

(6

marks)

3. Briefly discuss any four of the following; (5 marks each for any four = 20

marks) a. Evasion of phagocytosis by microbes.

b. Cytokines.

c. Major characteristics of an immunogen.

d. T-cell maturation in the thymus

e. Antibody production by B-cells

f. Immunoprophylaxis

SECTION B

4. a. What are hypersensitivity reactions? (2

marks) b. The ABO blood group incompatibility results in complement lysis of erythrocytes by

what type of antibody?

(1 mark) c. Explain what gives rise to contact hypersensitivity. (3

marks) d. Mention the immune system cells responsible for graft rejection. (3

marks) e. What is the manifestation of the chronic rejection of the following solid grafts:

47

Heart, kidney, lungs, liver. What factors increase the risk of this rejection? (9

marks) f. How can immune tolerance be induced in a prospective heart transplant recipient? (2

marks)

5. a. What is congenital immunodeficiency? (2

marks) b. What is the cause of the following immunodeficiency disorders?

i) DiGeorge syndrome (2

marks) ii) Chronic granulomatous disease (2

marks) iii) Bruton’s syndrome (2

marks) c. What is the effect of aging and Protein-calorie malnutrition on the immune system? (4

marks) d. Mention the factors that contribute to autoimmunity. (3

marks) e. List the symptoms of Hashimoto’s thyroiditis? (5

marks)

6. a. Why are serologic tests used to diagnose certain diseases? (2

marks) b. What are the manifestations of type III hypersensitivity reactions? Mention the

antibodies involved.

(7 marks) c. What are the clinical features of systemic lupus erythematosus? (6

marks) d. Mention factors that contribute to secondary immunodeficiency. (5

marks)