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SBTB031 Practical manual 2022

Industrial biotechnology practical manual 2022

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industrial biotechnology (sbtb031)

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University of Limpopo

Academic year: 2021/2022

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University of Limpopo

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Faculty of Science and Agriculture

SCHOOL OF MOLECULAR AND LIFE SCIENCES

Department of Biochemistry, Microbiology and Biotechnology

SBTB

PRACTICAL MANUAL

2022

Prepared by: Ms M Lekganyane and Mr L Ramoba

PRACTICAL MODULE SCHEDULES 2022

DATES TASKS

27-01-2022 Welcoming students and laying rules for the

module

03-02-2022 Introduction to laboratory skills, report writing and

laboratory safety

10-02-2022 Substrate Induced Production of an Industrial

Enzyme –preparation of inoculum

17-02-2022 Substrate Induced Production of an Industrial

Enzyme–submerged cultures

24-02-2022 Substrate Induced Production of an Industrial

Enzyme–lipase assay

03-03-2022 to

17-03-

Yogurt and wine production

24-03-

Beer brewing

07-04-2022 Practical test

Practical Session 1

In addition to observing the preceding basic requirements, the following specific

regulations for a teaching laboratory should be observed for the safety and

convenience of everyone working in it.

1. Street outerwear should not be brought into the laboratory.

2. Begin every laboratory period by disinfecting, or washing down, your bench-

top, and end it the same way.

3. If a culture is spilled, cover the area with disinfectant and notify the instructor.

Report all accidents, however minor, to the instructor.

4. Never remove any cultures from the laboratory.

5. Inoculated media placed in the incubator must be properly labelled with your

name, date, and nature of the specimen.

6. Pencils, labels, or any other materials should never be placed in your mouth.

7. Be very careful with gas burners. Long hairs should be tied back neatly away

from shoulders. Gas burners must be turned down or off when not in used

during exercises. Be sure gas burners are turned off at the end of the lab

period.

8. All reagents and equipment must be returned to their proper place at the end

of the lad period.

9. All used cultures and contaminated glassware should be put into a designed

container to be autoclaved.

10 plastic and other disposables are to be discarded into a

separate container also to be autoclaved.

11. Uncontaminated materials like scraps of paper and cotton must be placed into

wastebaskets.

12 lay contaminated materials like pipettes on the bench-top.

13 discard contaminated liquids or liquid cultures into the sink for disposal.

Put them into designated containers in the lab for later sterilization.

14 conduct in a biotechnology/microbiology lab should always be

courteous and professional. Personal attention to the principles of safety is

always in order.

It is your responsibility to make sure that you read and understand these

regulations. They are very important for your safety and the safety of other

users of the laboratory. Where you do not understand, ask your instructor

and s/he will explain further.

Guidelines for Writing Scientific Reports

Dept. of Biochemistry, Microbiology & Biotechnology

Summarise the results and
State the principal conclusions The conclusions must be stated three times, in the abstract, in the introduction and in the discussion. The abstract should always be the last to be written, because it is a summary of the report or document. This does not mean that it should be presented at the end of the report. It must still be in the beginning of the report.

The Introduction

The introduction should supply sufficient background information to allow the reader to understand and evaluate the results of the present study without the need to refer to previous publications on the topic. A good introduction must comply with the following rules: ) It should present firstly, with clarity, the nature and scope of the problem investigated or to be investigated. ii) To orient the reader to the relevant literature must be reviewed. iii) The method of investigation must be stated. iv) The principal results of the investigation should be briefly stated.

However, with specific reference to your experiments (iii) and (iv) must focus on the key principles of each scientific technique introduced and the importance of your key finding with respect to the information reviewed (ii). Information sources used/quoted in the introduction and discussion must be properly referenced, (see reference methods below).

Materials and Methods

The main purpose of this section is to provide enough detail so that a competent worker can repeat the experiments. Great care should be taken in

writing this section because the cornerstone of the scientific writing method requires that your results must be reproducible and for the results to be considered reproducible you must provide the basis for repetition of the experiments by others.

Materials Materials should include the exact technical specifications and quantities and source or method of preparation. Where necessary the chemical and physical properties of the reagents used must be stated. Experimental animals, plants and microorganisms should be identified accurately by genus, species and strain designations.

Methods Methods must be presented in chronological order. Related methods however should be described together.

Headings

Whenever necessary subheadings must be used. When possible subheadings should be constructed to match those used in the results section. Measurement and analysis Always state the conditions of incubation of your reactions i. temperature and period of incubation. Questions like How? and How much? Should be precisely answered.

Results

This section serves two objectives; a) First an overall description of the experiments should be given, taking care not to repeat the details provided in materials and methods.

) Try and present the principles, relationships, and generalizations shown by the results (4). You should never recapitulate the results. ii) Point out any exceptions or lack of correlation and define unsettled points. iii) Show how your results and contrast or agree with previously published work. iv) Discuss the theoretical implications of your work and possible practical applications. v) State your conclusions as clearly as possible. vi) Summarize you evidence for each conclusion. NB: When your results differ from what is generally expected, it usually reflects flaws in your technique; as a result, you will lose marks for lack of good technique. In the discussion section, you have an opportunity to redeem yourself by showing that you understood what you set out to achieve and what was expected by clearly identifying potential problems that led to failure of your experiment.

References

Where facts stated were obtained from a source the source must be properly identified and acknowledged. Different formats are used in referencing. You must use the following formats; ) The name date system

) Where only one author is concerned; e. Mbewe (1995) found that ............. Alternatively, state fact then reference, ........... Mbewe (1995) Where one author published two or more articles in the same year, which are both/all quoted. Either of the above approaches can be used but the years can be distinguished using letters, e. ............ Mbewe (1995a) Another reference could be made to another publication of the same year as follows, ............. Mbewe (1995b)

) Where two or more authors are involved. e. g. Mbewe and Moganedi (1999) found that............ Alternatively state fact and reference as follows; ......... Mbewe and Moganedi (1999).  The issue of the same authors having published more than once in the year and their articles being used is handled as above using letters after the year, i. ......... Mbewe and Moganedi (1999a) ......... .Mbewe and Moganedi (1999b)

c) Where more than two authors are involved. e. Instead of ......... Mbewe, Moganedi, Crous and Howard (1999) you should write ........ Mbewe et al. (1999).

) Listing references. The appropriate format must be used for listing references at the end of your report. The list of references must appear in alphabetical order. The following formats must be used depending on the nature of the source/reference.

) Journal articles;

 Prepare a starter culture or inoculum of Aspergillus niger.  Prepare media containing glucose and olive oil as carbon sources.  Grow A. niger in submerged cultures suitable for induction of lipase production.  Establish the optimum period for lipase production under these conditions.

1. Introduction

In industrial processes and products that apply enzymes, the source of the enzyme is usually a microorganism. However, microorganisms do not produce most of these industrial enzymes constitutively (all the time). The enzymes are produced when the microorganism detects a substrate/compound that it can use for survival. Hence, we consider these enzymes as having been induced by the substrate. Thus in industrial production of an enzyme, an inducer substrate/compound needs to be added for maximum production.

Most of the industrial enzymes are enzymes that degrade compounds utilised as carbon sources by the organism. Examples are amylases for starch utilisation, lipases for vegetable oils utilisation and cellulases for cellulose utilisation. Thus to produce these enzymes for use in industrial biotechnology process the organism has to be cultured in the presence of the substrates as carbon sources. Aspergillus niger is a fungus that produces a variety of enzymes. Hence it is able to grow on a variety of carbon sources. In industry, this mould is exploited for production of several enzymes under optimal conditions. In these experiments an optimisation of lipase production by the fungus A. niger is going to be done.

2. Materials

Viable Aspergillus niger Potato Dextrose Agar (PDA) plates 250 ml Erlenmeyer flasks Eppendorf tubes Olive oil

Glucose Tween 80 Mendels Media (per litre add the following :) K 2 HPO 4 2 g KCL 0 g FeSO 4 0 g MgSO 4 .7H 2 O 0 g KH 2 PO 4 7 g (NH 4 ) 2 SO 4 1 g Yeast extract 1 g Adjust pH to 6 before autoclave. After autoclaving add 5 ml of chloramphenicol (20 μg/ml) Isopropanol Para-Nitrophenyl palmitate Para–nitrophenol 0 M acetate buffer pH 6. Flat bottom 96 well micro-titre plates

2. Experimental methods

2 Preparation of Inoculum (Session 2)

Use a sterile surgical blade to cut an agar block of the organism from the given Aspergillus niger plate.

Place that agar block onto a Potato Dextrose Agar plate.

Incubate the plate at 30°C for 7 days.

2 Submerged Cultures (Session 3)

Use a sterile surgical blade to cut an agar block of A. niger from the PDA plate you inoculated the past week.

Practical Session 5

YOGURT PRODUCTION

Experimental objectives  to demonstrate the use of microorganismsfor industrial purposes  to introduce the yoghurt making process to students.  to demonstrate the microbiological activity of microorganisms involved in yoghurt production.

 to do a microscopic examination of the microorganisms involved in yoghurt production.

Introduction

Yogurt is a fermented milk product having a semiliquid consistency, produced by the action of lactic acid bacteria. It is manufactured from whole or skim milk, in which the solid content has been increased by the removal of water or the addition of powdered milk. The important starter organisms are Lactobacillus bulgaricus or Streptococcus thermophilus. These microorganisms produce lactic acid which causes the milk to be sour, by the following reaction: Lactobacilli Lactose Lactic acid.

The Gram positive Lactobacillus in yogurt are believed to exert a beneficial effect on the intestinal flora. The tart flavour of yogurt is attributed to the acetaldehyde but mostly the fruit flavouring is added to mask the tartness.

Materials

Microorganisms Media Reagents  Live culture of commercial yoghurt

 Dried powdered milk  Whole milk

 Methylene blue stain  Gram stain  pH indicator Strips

YOGURT PRODUCTION

Make slide preparations of your stater culture and the yoghurt you prepared (your own product after it has fermented). Stain with methylene blue and Gram stain, and examine under the microscope. Describe and draw the microorganisms observed and suggest possible names of spp. How do they compare the those you observed in part above.

Starter culture microorganisms Laboratory yogurt microorganisms

Possible identities Possible identities

Would you expect the identities of the microorganisms in both yogurt products to be similar or dissimilar? Give reasons for your answer. How would you explain dissimilarities?

 Check the pH of your product (the yoghurt). How does it compare to the pH of the fresh milk and your stater culture yoghurt? Explain your observations. pH of products Milk Starter yogurt Lab yogurt

 Examine the product and record the colour, taste, texture and aroma.

Description Starter yogurt lab yogurt

Colour Taste Texture Aroma

Practical Session 6

Wine Production

Experimental Objectives  to introduce students to the wine making process.  to demonstrate the microbial activity of microorganisms involved in wine production.  to evaluate chemical changes in wine during fermentation process  to determine the organoleptic properties of produced through sensory evaluation.

Introduction

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