Spills
Spills should always be cleared up immediately. While a few may need absorption and/or chemical neutralization using a spill kit (or disinfection or similar treatment), most minor spills can be dealt with by a damp cloth. For large spills of chemicals producing hazardous fumes, there may be a need to call the fire brigade. Students should be encouraged to report spills and breakages, so that they can be cleared up immediately, and not left to cause injury to the next class, or to a technician or cleaner.
Personal cleanliness
Students should always wash their hands after practical work with chemicals or with soil or material of living origin and facilities should permit this.
Broken glass
Glass vessels, such as test tubes, flasks and beakers, should be checked for cracks and chips before use. Particular care should be taken when glass containers are evacuated; use round-bottomed or pear-shaped vessels, check for cracks, and protect observers by one or more safety screens. Broken glassware should be placed in a specially labeled bucket. It should be wrapped in newspaper or sealed in a non-perforatable container, such as a box or metal can, before disposal with normal waste.
Handling glass
When inserting corks, stoppers or bungs into test tubes or specimen tubes, pipettes into safety fillers or glass tubing or thermometers into bungs, students need to be shown a safe technique (see Handbook or Safeguards). Inserting glass tubing or thermometers into bungs is generally best left to technicians, who may themselves need training.
Many different types of nutrients can be added to agar. Plan and set up a practical, which will investigate the effect of growing a micro-organism on agar containing different nutrients.
There are two types of media –
- Broth - this is a liquid
- Agar – This is the same as broth, but it contains a jelly, which sets the broth (note: the jelly has no nutritional value.)
Experiment:
The experiment was to discover which nutrient agar was best suited to growing fungi. Three types of nutrient medias were used:
To grow the fungi, three types of nutrient agar are used:
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Malt Agar (MA): lacks peptone, and is useful for culturing many Ascomycota; sporulation in some species is inhibited by peptone.
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Potato Dextrose Agar (PDA): a relatively rich medium for growing a wide range of fungi.
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Czapec-Doz agar Elective agar proposed by CZAPEK (1902-1903) and DOX (1910) for the cultivation of fungi and soil bacteria. This culture medium contains sucrose as its sole carbon source and nitrate as its sole nitrogen source.
When these are made, the ingredients are mixed with water then autoclaved, to kill any micro-organisms that may be present.
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Decide on the type of media - Agar
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Decide how much yeast/concentration of yeast cells - x10 -2
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Temperature - Incubator is 30oc
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Time period for growth -1-2 days
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Container material -Plastic/circular
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Plan -Step by step guide
- Measurement of growth
- Data presentation
It is important that this experiment is carried out using the aseptic technique so that the samples do not become contaminated.
Step one: Collect equipment; three petris containing the required media, inoculating loop, alcohol, cotton wool, Bunsen burner and lab coat.
Step two: Put on the lab coat, sterilise the working area using the alcohol and cotton wool, so that any micro-organisms will be destroyed
Step three: The yeast needs to be diluted to the require concentration for the experiment. For this serial dilution is used:
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A dot of yeast is stirred into 1cm3 of water and then diluted further with another 9cm3 of water into a test tube.
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1cm3 of this dilution is then removed and further diluted with 9cm3 of water into another test tube.
However the lab technicians carried out this part of the experiment.
Step four: After lighting the Bunsen burner, hold the inoculating loop in the blue flame until it glows red, this will eradicate any micro-organisms that may be present. Be sure to turn the Bunsen burner off after you have finished using it.
Step five: Carefully lift off the lid of the petri dish, making sure you only remove is sufficiently to gain access. Be careful not to keep the petri lid of for to long, or to let your fingers touch the media, as this will introduce unwanted micro-organisms to the media:
Dip the sterile inoculating loop into the sample, then making quick stokes spread the sample on the media.
Step six: The samples are placed in the incubator for 1-2 days
Results:
We were quite disappointed with the results, as they did not tell us what we thought they would, Czapec-Dox agar is the recommended media for growing yeast, however in this case the results are disappointing as the observable growth, and what growth there was, was barely visible a tall on this media was minimal at an estimated coverage of 2.9%.
The Potato Dextrose Agar was more encouraging as there was more noticeable growth and the estimates coverage was 8.6%.
The Malt Agar turned out to be the best media as the visible growth was highly evident and the estimated coverage was 10.2%
How this is calculated: Each numbered box is looked at and the percentage the square that is covered with cells is estimated. For example in the picture below, this square has about 25% coverage. Once all the square have been estimated the total is added together:
Once a pure culture of bacteria has been obtained the bacteria may be mounted on a microscope slide and identified using gram stain. Bacteria are either gram negative or gram positive.
Approximately one third of patients admitted to hospitals have, or develop, infections, many of which are caused by bacteria and fungi.
In most cases, the organisms cannot be identified by clinical symptoms. a Gram-stained smear specimen is obtained and often gives important information that is not obtained from a culture of the specimen.
Some bacteria only weakly stains or not at all with the Gram staining technique. This requires a special staining technique. Such as acid-fast staining for mycobacteria and nocardia, and immunofluorescent stain for Legionella.
The Gram stain procedure was originally developed by the Danish physician Hans Christian Gram to differentiate pneumococci from Klebsiella pneumonia.
The procedure entails adding a solution of iodine to cells that have previously been stained with crystal violet or gentian violet. This procedure produces "purple coloured iodine-dye complexes" in the cytoplasm of the bacteria.
The cells that were previously stained with crystal violet and iodine are next treated with a decolonising agent; 95% ethanol or a mixture of acetone and alcohol.
The difference between Gram-positive and Gram-negative bacteria is in the permeability of the cell wall to these "purple coloured iodine-dye complexes" when treated with the decolourising solvent.
Gram-positive bacteria keep the purple iodine-dye complexes after treatment with the decolourising agent; Gram-negative bacteria do not retain complexes when decolourised.
To visualize decolourised Gram-negative bacteria, a red counter-stain such as safranin is used after decolourisation.
The right preparation of the smear sample is essential.
Make a thin film of the sample on a clean glass slide, using a sterile loop. Air dry and then heat fix the slide by passing it several times through a flame (the slide should not become too hot to touch).
To be visible on a slide, organisms that stain by the Gram method must be present in concentrations of a minimum of 104 to 105 organisms/ml of unconcentrated staining fluid. At lower concentrations, the Gram stain of a clinical specimen seldom reveals organisms even if the culture is positive. Smears that are not properly fixed tend to be washed away during staining and washing resulting in the absence of stained bacteria.
Source: www.meddean.luc.edu
Micro-organisms are extremely useful in many areas of our lives, they are used to produce enzymes, to make medicines and vaccines and in bread and beer production. To be used in manufacturing industries the microbes have to be grown on a large scale and in industrial fermenters or bioreactors.
The conditions inside a fermenter have to be monitored and controlled so that maximum growth of the micro-organism is achieved. Explain how ideal conditions inside the fermenter are maintained.
A fermenter is a large stainless steel tank, that is used to grow manufactured micro-organisms in the industrial production of enzymes, yeast, vitamin C and penicillin and other micro-organisms. After the tank is sterilized, an amount of the producer cells is introduced into a medium that is maintained by probes at optimal conditions of temperature, pressure, pH, and oxygen levels for the most advantageous production.
An agitator mixes the medium, which is constantly aerated. It is essential that the culture medium is sterile and contains the correct nutritional requirements for the micro-organisms.
When the nutrients have been utilized, the products are removed, if the product is an extracellular compound, the medium can be removed during the growth phase of the micro-organisms, but an intracellular product must be collected when the batch culture growth stops.
One method of monitoring the growth of the microbes is to take samples and count the number of cells. This can be done using a haemocytometer or by growing the microbes on plates and counting the colonies. You will perform both of these counting methods and calculate the number of cells.
Haemocytometers, originally designed for counting blood cells, can also be used for counting cells of bacteria, yeast or algae. At the centre of a haemocytometer is one or more grid that is overlaid by a specially designed cover glass. The design is such that when a cell suspension is placed between the cover glass and the grid, the haemocytometer retains a unit volume of liquid above each square. After placing a small drop of cell suspension in position with a dropping pipette, and replacing the cover glass, the grid is viewed under a microscope and the cells are counted.
Equipment
Suspension Of Yeast Cells
Haemocytometer
Dropping Pipette
Microscope (x15 eyepiece and x10 and x40 objectives)
Cloth, Moistened With 95% Alcohol
Method
If the haemocytometer has two grids, separated by an H-shaped drainage well.
Place a small drop of the sample above the grids. Place the cover glass and allow any excess liquid to drain into the well.
Transfer the haemocytometer to the stage of the microscope.
Focus on the squared region
To calculate the number of cells in 1cm3 of the suspension, Each of the 25 squared units measures 0.2 x 0.2 x 0.1mm (depth), and has a volume of 0.004mm3. After counting the number of cells in one of the 25 squared units, (multiplied by 250000 to give the approximate number of cells in 1cm3 of the cell suspension).
See attached diagram
References
Books:
Brooker.C., Human Structure and Function (se), Mosby, 1998, London
Marieb.E., Human anatomy and physiology (se), Benjamin Cummings,1992, USA
Roberts. J., Mastering Human Biology, Macmillan 1991
Journals:
N/A
Websites:
www.ase.org.uk/safety
www.biosci.org.uk/misac
www.cf.ac.uk
www.microbiologyonline.org.uk
www.parliament.the-stationery-office.co.uk
www.sciencefirst.8m.net
APPENDICES:
Government legislation
ENVIRONMENTAL SAFETY
35. The competence of the European Community to legislate on biotechnological matters is based on a number of Treaty provisions covering the environment, health and safety of workers, and the approximation of laws to establish an internal market. There are, in addition, regulations in place that apply to the agricultural use of all products, whether genetically modified or not. Micro-organisms modified in the research or development laboratory are subject to regulation under the terms of the contained use Directive (90/219/EEC) and the Biological Agents at Work Directive (90/679/EEC). The latter applies only to micro-organisms, but the definition includes animal and plant cells in tissue culture. There are specific United Kingdom regulations governing the use and treatment of animals.
36. If there is an expectation that the organisms may be deliberately released into the environment, Part B of the deliberate release Directive (90/220/EEC) imposes a notification requirement and specifies the information which must be provided to the competent national authorities. This comprises a technical dossier and an evaluation of the impact and risks to human health or the environment. Part C of the Directive establishes a Community procedure for authorising consents for marketing genetically modified organisms (whether released into the environment or not). This includes a requirement for an environmental risk assessment, except if such or a similar assessment is mandatory in respect of products covered by other EC legislation. The Commission's proposed revision of Directive 90/220/EEC was published on 26 February 1998, and it is on this text that we base our specific comments on the reform of the regulatory system.
MICRO-ORGANISMS FOR INVESTIGATIONS IN
SCHOOLS & COLLEGES
In May 1997, a safety conference was convened by the Association for Science Education. Various
organisations were represented, including the ASE, CLEAPSS, SSERC, HSE, MISAC (Microbiology in
Schools Advisory Committee), Society for Applied Microbiology, Society for General Microbiology,
NCBE (National Centre for Biotechnology Education), SAPS (Science & Plants in Schools), the
Wellcome Trust and the educational suppliers Philip Harris and Blades Biological. The principal aims of
the conference were to consider clarification of guidance on the use of micro-organisms and
biotechnology in the DfEE publication Safety in Science Education and to evaluate the list of microorganisms
considered suitable for use in schools and colleges following changes to the hazard categorization of certain micro-organisms by the Advisory Committee on Dangerous Pathogens1. One of the outcomes of the conference is a revision of this list. The accompanying tables give selected micro-organisms which present minimum risk given good practice. These tables supersede the existing lists found in the CLEAPSS Laboratory Handbook (1992), the CLEAPSS Shorter Laboratory Handbook (2000), Microbiology: An HMI Guide for Schools and Further Education (1990, now out of print), Topics in Safety (1988) and Safety in Science Education (1996).
As well as naming suitable organisms, the new lists give points of educational use and interest and comment on the ease with which organisms can be cultured and maintained. The lists of microorganisms are not definitive; other organisms may be used if competent advice is obtained.
It should be noted that strains of micro-organisms can differ physiologically and therefore may not give expected results. Where possible, fungi that produce large numbers of air-borne spores should be handled before sporulation occurs, so that the spread of spores into the air and possible risks of allergy or the triggering of asthmatic attacks are minimized. This is particularly important for some species, such as Aspergillus and Penicillium, which produce very large numbers of easily dispersed spores. It should be noted that certain species of these two fungi, previously listed as unsuitable for use in schools, are now not thought to present such a serious risk to health, given good practice in culture and handling.
Inoculation of cultures
Inoculation should involve precautions to prevent contamination of the person and work surfaces. It should also avoid the contamination of culture media with unwanted microbes. Media and Petri dishes etc. should either be purchased pre-sterilized or sterilized by the user before agar plates are poured.
Media must not be deliberately inoculated with material likely to be sources of human pathogens.
L3 For the aseptic transfer of cultures, work surfaces should be swabbed with a suitable disinfectant before and after all operations and sufficient time allowed for disinfection to occur. Arrangements should be made to sterilize inoculating loops and spreaders before and after inoculation, and to provide discard pots for pipettes and syringes. The mouths of all containers, tubes, flasks, McCartney bottles etc, should be flamed after removing caps and before their replacement. Lids of Petri dishes should be opened only just enough to allow the inoculating tool to be introduced and for as little time as possible.
Bench surfaces For practical work by students, benches should be wiped down with a cloth soaked in a suitable disinfectant, preferably before, but always after practical work, and sufficient time allowed for disinfection to occur.
Observation of cultures of bacteria and fungi
L1 Cultures should be viewed in the unopened containers in which they were grown.
L2 Cultures should be examined by pupils in containers, which have been taped closed. If cultures may contain pathogens and there is a risk that students may open them, even though instructed not to do so, it will be prudent for the cultures to be completely sealed with tape before examination. If it is necessary for pupils to open cultures for examination, special precautions may be necessary. Other than for pure, non-pathogenic cultures prepared by teachers or technicians under aseptic conditions, and especially for cultures originating from environmental samples in which pathogens may be present, these must be killed by the teacher or technician as follows. A filter paper is placed in the lid of an inverted agar plate and moistened with 40% methanol solution (formalin). After 24 hours the filter paper is removed. (Take care with methanal: eye protection, gloves and use of a fume cupboard to avoid breathing fumes are necessary).
L3 Cultures of known and non-pathogenic microbes can be examined using a variety of techniques. Organisms cultured from body surfaces or any environmental source must be examined in unopened containers, or killed before examination as described above. Sterilization and disposal. All cultures must be heated to kill micro-organisms before disposal. This is best done using a pressure cooker
or autoclave, in conjunction with autoclavable bags. The caps of all screw-topped bottles must be loosened before cultures and media are sterilized. It is very important that instructions for use of the autoclave are followed in order to achieve and maintain sufficiently high temperatures for a long enough time. Pressure cookers are unlikely to be equipped with appropriate instructions for sterilization and those for some autoclaves, designed for use with surgical instruments, state that the equipment is unsuitable for sterilizing liquid media. Such autoclaves can be used for microbiological preparations but advice on their correct operation should be sought. Teachers and technicians should be trained to follow safe working practices. Seals and safety valves should be checked before each use. Heating autoclaves or pressure cookers with Bunsen burners is not recommended. Rapid cooling and the release of steam to lower the internal pressure quickly to atmospheric pressure is dangerous because it may shatter glassware and/or cause liquid media to boil over. Equipment should be allowed to cool unaided before opening. Further information may be sought from the advisory bodies such as MISAC. Sterilization cannot be achieved by the use of chemical disinfectants. If, in exceptional circumstances, chemical disinfection of cultures is contemplated prior to disposal, use a freshly-made solution of a disinfectant that is not degraded when in contact with organic matter (see ‘Spills’ above). Cultures and equipment must be opened under the surface of the solution and left for at least 12 hours. Again it is essential to follow disinfectant instructions carefully. Chlorate(I) solution is inactivated by large amounts of organic matter, although if a culture might contain viruses this is often the preferred disinfectant.
BTEC National Diploma Health Studies
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