MCB 203: Laboratory Methods in Microbiology 2 Credits• OUTLINE

• Introduction ( Microbiological procedure & list of equipments)

• Culturing of microorganisms• Preparation of media for microbial growth. Isolation of

pure culture; streaking, pour plates, sub-culturing procedures.

• Staining techniques for differentiation of microorganisms.• Enumeration of microorganisms, direct and indirect

procedures.• Identification of microorganisms to include colonial and

cellular morphology and biochemical procedures. Microscopy

• Sterilization techniques• Preparation of microscopic slides.

INTRODUCTIONIn any laboratory set up for diagnostics purposes, there should be a well arranged system that must be put in place to ensure good results, such system must ensure good laboratory practice, proper handling of equipment, sampling and sample preparation/ reagents, proper documentation of the data obtained.

This lecture will introduce us to some basic diagnostic equipment, method of handling in order to ensure good results and its durability.

Also we will understand how to prepare samples for analysis, data recording and extraction of samples for detection of pathogenic organisms from plants

INTRODUCTION TO LABORATORY EQUIPMENT

An autoclave is a device to sterilize equipment and reagents, and media by subjecting them to high pressure steam at 121° C or more. There are two main types of Autoclaves:Stove top autoclaves They resemble a pressure cooker. Each unit comes complete with a bolt-down lid and a pressure gauge on the outside. Front loading autoclaves are more widely used for their convenience, The units are box-shaped and self contained, equipped with a heating unit to turn water into vapour for sterilization.. There is also a gauge to track chamber temperature/pressure

CARE AND MAINTENANCEAutoclave requires more caution and must be handled with careEnsure that the water covers the ringRemove the plug from main source when not in useWash the autoclave in case media or reagents pour into the autoclaveEnsure there are no leakages during the process of sterilisation.

LAMINAR FLOW CHAMBER

The vertical hood, also known as a biology safety cabinet, is best for working with hazardous organisms since the aerosols that are generated in the hood are filtered out before they are released into the surrounding environment. Horizontal hoods are designed such that the air flows directly at the operator hence they are not useful for working with hazardous organisms but are the best protection for your cultures.

Both types of hoods have continuous displacement of air that passes through a HEPA (high efficiency particle) filter that removes particulates from the air. In a vertical hood, the filtered air blows down from the top of the cabinet; in a horizontal hood, the filtered air blows out at the operator in a horizontal fashion.

NOTE: These are not fume hoods and should not be used for volatile or explosive chemicals. They should also never be used for bacterial or fungal work.

CARE

The hoods are equipped with a short-wave UV light that can sterilize the surfaces of the hood.

Do not put your hands or face near the hood when the UV light is on as the short wave light can cause skin and eye damage.

The hoods should be turned on about 10-20 minutes before being used.

Wipe down all surfaces with ethanol before and after each use.

Keep the hood as free of junks as possible because this will interfere with the laminar flow air pattern.

Microscope is an essential equipment for identification of Pathogenic organisms such as Mould and BacteriaIt helps to study The Internal structures, Morphological structures and Reaction to stains

CARE OF THE MICROSCOPE

STORAGE:- When not in use for a long time, keep the microscope in a box with hydroscopic silica gel to prevent mould on the lenses or cover with dust proof material such as polytheneTurn Lp objective into position and not HP objective lensDo not remove the objective lenses from the nosepiece

CARRYING A MICROSCOPEWhen carrying a microscope out side the box hold the arm of the microscope in one hand and support the base with the other hand

Carry the microscope in an erect position lest the eye piece drops out of its tubes

Do not clean the lens with toilet paper

Use a lint free handkerchief to clean the main body but not the lenses or use camel air brush

Do not use hand to clean the lens.

It involves the use of the centrifugal force for the separation of mixtures, used in industry and in laboratory settings. More-dense components of the mixture migrate away from the axis of the centrifuge, while less-dense components of the mixture migrate towards the axis.

Molecular Biologists may increase the effective gravitational force on a test tube so as to more rapidly and completely cause the precipitate ("pellet") to gather on the bottom of the tube. The remaining solution is properly called the "supernate" or "supernatant liquid". The supernatant liquid is then either quickly decanted from the tube without disturbing the precipitate, or withdrawn with a Pasteur pipette.

CARE

The load in a laboratory centrifuge must be carefully balanced. This is achieved by using a combination of samples and balance tubes which all have the same weight.

Small differences in mass of the load can result in a large force imbalance when the rotor is at high speed. This force imbalance strains the spindle and may result in damage to the centrifuge or personal injury.

Some centrifuges have an automatic rotor imbalance detection feature that immediately discontinues the run when an imbalance is detected.

Before starting a centrifuge, an accurate check of the rotor and lid locking mechanisms is mandatory. Centrifuge rotors should never be touched while moving, because a spinning rotor can cause serious injury. Modern centrifuges generally have features that prevent accidental contact with a moving rotor as the main lid is locked during the run.

Wash in case there is split of sample inside the rotor

Unplug the equipment from electrical source

It is used for enzyme assay Maintaining Media at certain temperature before pouringPreparation of media such as Salmonella Shigella agar

•Test for Aflatoxin in food sample to ensure its safety for consumption• Detection of infection in Plant sample•Study enzyme activity

SAFE HANDLING/ CARE AND MAINTENANCE

Unplug the equipment when not in use

Cover the equipment to avoid dust settling on it

Ensure that the Plate is well seated on the plate frame before reading( do not force)

APPLICATION•Detection of Pathogen in infected plants such as Fungi, Bacteria and Virus

SAFE HANDLING/CARE AND MAINTENANCE

Unplug the equipment when not in use

Do not run the equipment unattended

Keep it away from water

Use power supply with safety features that can detect any leakages or no load and overload

When connecting wear protective glove

Any leakages of water should quickly attended to

Avoid dust

Maintain the equipment below 370C

It is used for accurate measurement of chemicals Measurement of sample

SAFE HANDLING/CARE AND MAINTENANCE

Place balance in an air controlled room

Do not carry around

Do not leave the material weighed on balance for a long time

Avoid spillage

Do not use filter paper to weigh

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