The reported notes are available on the web www.fluorescence-Microscope.com

In fluorescence microscopy, fluorophores are used to reflect an image of a given sample or specimen. A fluorescence microscope is generally made up of a specialized light source, either Mercury or Xenon, excitation and emission filters, and a dichroic mirror. The following steps will instruct you how to use a fluorescence microscope properly and safely.

Step 1: Remove the protective cover of your fluorescence microscope. Make sure it is set at low power before plugging and switching it on. Turn on the mercury lamp as well. You will have to wait approximately fifteen minutes before the microscope can provide full brightness. Switch on the motorized focus box.

Step 2: Place your prepared slide properly on the stage. Secure the slide with the stage clips. Peek through the eyepiece and slowly make the necessary adjustments to bring your sample into focus. The coarse knobs are there to lift or lower the stage while the fine knobs are there to provide a sharper and clearer image of your specimen.

If you are switching to a different objective, do so by holding the collar of your fluoresce microscope’s nosepiece. Never exert pressure on the objective lenses themselves because this could cause them to lose their alignment.

Change of filters is best performed while the microscope is set at low power. If you are going to adjust the condenser for Kohler illumination, refrain from adjusting the stage knobs.

Step 3: If you are interested in taking pictures of the sample, you can attach a camera eyepiece to the microscope. Images will be stored on your camera’s built-in memory or on a connected storage device.

Step 4: If you are finished using the microscope, you can only switch off the microscope if you have consumed at least thirty minutes. If so, remove the slide from the microscope then switch off the focus control box. Switch off the mercury lamp. You can turn on the fluorescence microscope again after half an hour.

Step 5: Unplug the fluorescence microscope and return it to its original place and under its protective cover.

Other Tips When Using a Fluorescence Microscope

- Mercury lamps release extremely potent and visible UV radiation so avoid looking at them directly. Do not disassemble the lamp housing. Do not look into the fluorescence microscope’s eyepieces when you are changing filters. Certain filters can reflect the UV rays directly to your eyes.
  
- Always record how many hours you’ve used the mercury lamp of a fluorescence microscope. Going beyond its expected lifespan can put your fluorescence microscope at risk of exploding.
  
- Switching a mercury lamp on and off frequently can reduce its lifespan. Leave it on if you expect someone else to use the fluorescence microscope in two hours’ time.
  
- If you are going to use oil immersion objectives for your fluorescence microscope, be careful when you are using low power objectives. Oil from your slide could contaminate your objectives when you swing the microscope’s nosepiece the wrong way. After using them, you can clean the lenses with lens paper by dabbing off the oil. Do not wipe harshly.
  

Using Epifluorescent Illumination with Your Fluorescence Microscope

Step 1: Place the appropriate filter. If you are going to use an epi-polarization filter, make sure to switch off the mercury lamp first. You can only look in the eyepiece after replacing the filter.

You can use bright field or other techniques – reflected or transmitted – to focus on your sample. Switch on the Mercury lamp.

Place in the correct filter cube for your microscope’s fluorochrome. Check if the analyzer slider has been detached; if not, then signal intensity would be reduced.

Step 2: If the Nomarski prism is still inserted in the space on top of your microscope’s objective, remove it. Its presence only reduces the quality of images captured by your fluorescence microscope.

Step 3: Take out your microscope’s shutter slider and place it in the desired filter position.

Step 4: Push the control lever in to open up the condenser aperture of your fluorescence microscope. Another push would open the field aperture of your condenser.

Step 5: You can now start observing your specimen. Adjust what’s necessary. If you’re done, push the fluorescence shutter in. Forgetting to do so places your sample at risk of overexposure and consequently, photobleaching. In photobleaching, fluorophores lose their ability to “glow” when they are illuminated.

You now know how to use a fluorescence microscope. Make sure to have a copy of this article posted near your work table to avoid forgetting any of the important safety tips included here.

Source: Fluorescence-Microscopes.com

Proper sanitation and disinfection are important to any program of animal care. The Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals include provisions for appropriate sanitation and disinfection of animal quarters and equipment.  In this regard it is essential that the efficacy of disinfection procedures be monitored to assure adequate inactivation of potentially pathogenic microbes.

The Guide lists microbiologic monitoring as a direct way of measuring the efficacy of the disinfection of laboratory equipment.

Selecting a Biological Safety Cabinet, it is first of all important to understand the basic difference between a fume hood, an unidirectional (laminar) flow cabinet and a Biological Safety Cabinet.

The “fume hood” is designed to duct corrosive chemicals outside the building but it is not equipped with HEPA filters; therefore the microbiological agents inside a fume hood will be ducted outside, contaminating the environment.

The “unidirectional flow cabinet” provides only product protection by blowing clean sterile air across the work area inside the cabinet toward the operator. Therefore the microbiological agents present in the work area will invest the operator’s body.

The “Biological Safety Cabinet” is designed to protect the operator, the product, the environment by HEPA filtering the inlet and outlet air. The microbiological agents are always contained inside the cabinet.

Biosafety Levels

The “Biological Safety Cabinet” should be selected according to the degree of lethality and medium of transmission of the microbiological agents that are classified under four categories of Biosafety Level (“BSL”).

Biological Safety Cabinet Classification

The BSC are classified in three different classes:

“Class I Biological Safety Cabinet” has inflow that protects the operator, but not the product because the outside air will contact the sample inside the work area. This BSC is therefore not very popular in the microbiological laboratory.

“Class II Biological Safety Cabinet” has inflow to protect the operator, downflow HEPA filter to protect the product in the work area, HEPA filter to filter the exhaust.

“Class III Biological Safety Cabinet” has a completely closed front. The analytical manipulations are performed through integrated gloves. The BSC is hermetically sealed and the material is introduced and taken away from the work area thru a double-door pass box. The product is protected by a stream of HEPA filtered air. The work area is under negative pressure and the air is exhausted passing a HEPA filter. This BSC is indicated for Biosafety Level 4 micro-organisms.

Cytotoxic Safety Cabinet

The Cytotoxic Safety Cabinet (CSC) is required for chemotherapy drug preparation. Such CSC has a third HEPA filter to protect not only the operator during the normal daily activity, but also the servicing people during the exhausted HEPA filter replacement. In fact the cytotoxic drugs are very toxic and cannot be neutralized by formaldehyde or hydrogen peroxide decontamination.

Biosafety Cabinet Certification

The BSCs should be certified by an independent international agency according to standard like NSF 49:2002 or EN 12469:2000 to guarantee their performances.

The requested standard include 3 different tests:

(a) Operator protection

Bacteria are sprayed behind the front window and they should escape through the front opening.

(b) Product protection

Bacteria are sprayed in front of the window and they should not reach the work area.

(c) Absence of Cross contamination

Bacteria are sprayed inside the BSC at one side of the work area and they should not reach the adjacent area, 20 cm apart.

Sterilization by steam autoclaving is not correctly performed if only time and temperature parameters are considered. The following notes has the purpose to help the staff to perform a correct sterilization cycle.

1. Air trapping. Steam displaces air downward. Therefore, long, thin containers should not be placed in the autoclave in an upright position. Sterilization of materials in plastic bags will likewise fail, unless the bags are wide open and only partly filled to allow access of steam and escape of air.

2. Heat penetration. A cold object placed in an autoclave takes time (over and above the time it takes for the chamber to reach sterilizing temperature) to heat adequately. For example, a 1 liter container of fluid must be autoclaved for 25 minutes after the chamber reaches 121°C, but a 4-liter container may require an hour for sterilization.

3. Contact with steam or water. Dry microorganisms protected from contact with steam (for example, with oil, plastic wrappings, or containers of talcum powder) cannot reliably be killed by autoclaving.

4. Heat indicators. Test tubes and tapes containing a heat-sensitive chemical indicator are often included along with objects when they are autoclaved. The indicator changes color during the autoclaving and thus gives a visual means of checking whether the objects have been subjected to adequate heat. A changed indicator does not always indicate that the object is sterile, because heating may not have been uniform, and even stored wrapped objects may have organisms reintroduced (see No. 7).

Tubes or envelopes containing large numbers of heat-resistant spores of the nonpathogenic bacterium Bacillus stearothermophilus, which acts as a biological indicator, are also frequently included with packs of materials being autoclaved. Death of these spores indicates adequate killing at the point where the tube or envelope was placed, which should be near the center of the material being autoclaved.

5. Elevated boiling points under pressure. Fluids in autoclaves are prevented from boiling, even though well above their normal boiling point, by the elevated pressure. If at the end of the period of autoclaving, valves are opened to release the pressure, these fluids will immediately boil and may even explode their containers. The pressure must be maintained to prevent boiling until the temperature has dropped below the boiling point at atmospheric pressure.

6. Negative effects of heat on some materials. Most autoclaves can be adjusted to operate at temperatures lower than 121°C, the usual autoclaving temperature. The use of’ lower temperatures is satisfactory for materials that can be tested for successful sterilization before being used. For example, certain heat-sensitive bacteriological media are sterilized at 115°C. This temperature is usually effective because heat-resistant microbial contaminants are rarely present in the media. Moreover, samples of the autoclaved media are easily tested for sterility before being used.

7. Prevention of recontamination. Objects to be autoclaved are usually wrapped in paper or cloth to allow penetration of steam during sterilization and to prevent recontamination thereafter. When wet, these coverings are readily permeable to bacteria and should therefore be allowed to dry before removal from the autoclave chamber. After that they should be stored in a closed cupboard or drawer to prevent the reintroduction of contaminants by ants, moths, or other means.

References

E. Nester, E. Roberts, M. Lidstrom, N. Pearsall, M. Nester: Practical Aspects of Autoclave Use – Microbiology, Saunders College Publishing. pg 802-803.

The microwave oven is recommended for saving time during microbiological culture media preparation when it is necessary to have available melted media in a few minutes.

The microwaves cross, without heating, air, paper, wood, earth, china, glass, plastic material and are absorbed by the media and other organic and inorganic substances.

General recommendations

The microwaves are reflected from metal objects like steel, iron , aluminum , etc.; do not use metal containers o metal caps.

The door and air vents can become quite hot. Take care not to touch these areas.

To avoid damaging your oven, never operate it when empty.

If you leave the media in the oven too long, it will dry and burn.

Make sure that the container is suitable for use with a microwave oven. Do not use metal containers.

Installation

Position the oven on a flat , horizontal surface. Keep it clear of any source of heat or steam.

Check that air can flow freely around it. Leave sufficient space between the oven and the wall: 5 cm at the sides and at least 10 cm at the back.

Never lay anything on the oven which could block the ventilation orifices at its back.

Cleaning

A damp soapy sponge should be used to clean the inside and outside of the oven.

The periphery of the door and oven must always be clean.

It is not recommended to use abrasive products, alcohol or thinners which could damage the oven.

Hot vinegar is suitable for wall wiping.

Melting of agar media

a. Use glass Erlenmeyer flask o Pyrex bottle with plastic screw cap. Never use metal cap.

b. The cap must never be hermetically closed. It must be always unscrewed. An hermetic closed container could explode.

c. When the medium starts to liquify , open the oven door and mix the content of the glass container to obtain an uniform temperature inside the media.

d. When using small containers ( 25, 50, 100 ml ) include them in a bigger larger vessel containing water to obtain a more regular and progressive heating.

e. When using test tubes, include them in a Pyrex beaker containing water. Be sure the test tube caps are not made of metal ( plastic caps or cellulose plugs should be used ).

f. The heating time should be selected by experience using different amount of media.

Question 1

How is important the use of specialized anaerobic media?

Answer:
1. Media like Columbia Tryptic Soy, Wilkins Chalgren and Schaedler are unsuitable.
2. Two media are better that one because many anaerobic infections  contain more than one type of anaerobe.
3. Incubation should be for a minimum of 48 hours and continue until at least 5 days.
4. The common selective media:

Question 2

Which are the common reasons for the failure of anaerobiosis?

Answer:
- Anaerobe jar leakage
- Poor catalyst activity
- Failure to use biological and chemical indicators
- Wrong biological indicator
- Failure to include CO2 in the anaerobic atmosphere
- The so-called “five o’clock build up”: waiting until the end of the day before incubating anaerobic plates.

Question 3

Which are the common reasons for the failure of anaerobiosis?

Answer:
- Anaerobe jar leakage
- Poor catalyst activity
- Failure to use biological and chemical indicators
- Wrong biological indicator
- Failure to include CO2 in the anaerobic atmosphere
- The so-called “five o’clock build up”: waiting until the end of the day before incubating anaerobic plates.