EDCUR 327.3 How to...

EDCUR 327.3

Methods for Teaching Life Sciences in the Secondary School

How to...

Agar Plates

Janet McVittie

The basic recipe is 15 grams of agar powder to 1 litre of distilled water. The agar must be dissolved in hot distilled water. Boil water and pour it over the agar, then stir it until the agar is dissolved. Then you can put the container of agar in a pressure cooker to “autoclave” it thus killing any bacteria or fungi which might have fallen in. Sometimes you can skip this step. It is likely that most bacteria and fungal spores have been killed in the hot water. If it is important that the plates be absolutely sterile, you must autoclave in a pressure cooker.

However, you would not prepare media with just agar. You need some sort of nutrient to add to the media. Following these general descriptions are a few recipes for nutrients which can be added; the nutrients you add will affect the kind of organisms that grow. You can grow bacteria, fungi, algae with agar media.

Cool the agar to 50-60C before pouring the plates. This will reduce the condensation of water in the plate (http://cse.tnaes.affrc.go.jp/suzuki/chlamy-media.html). You do not want condensation because it becomes difficult to see the colonies growing. You normally store the plates upside down, so any condensation there is will not fall onto the growing colonies; however, the condensation interferes with your ability to see the colonies. When growing from a random sample, you do not want your students opening their plates. Some of the bacterial colonies might be unhealthy for us humans.

Nutrients

Euglena medium (http://cluster3.biosci.utexas.edu/research/utex/media/growth-notes.html)

Suitable for most cultures of Euglena.

Preparation: to 1000 mL of glass-distilled water, add:

sodium acetate 1.0 g

beef extract 1.0 g

tryptone 2.0 g

yeast extract 2.0 g

CaCl2.2H2O 0.01 g

Optional ingredients: agar at 15 g/L to solidify

Soilwater as a Medium (for those organisms which live in soil!)

(http://cluster3.biosci.utexas.edu/research/utex/media/growth-notes.html)

Soilwater Supernatant:

The supernatant of soilwater (GR+) medium is included as an ingredient of some media.

Preparation: In 500 mL beaker, combine 5 mL of Garden soil (dry), small amount of CaCO3, and 200 mL of distilled water.

Cover the beaker and steam for 2 h on each of 2 consecutive days. Refrigerate (10° C) 24 h or more and bring to room temperature before using. Decant the supernatant, and add the required amount to the medium.

Growing Cultures

For growing cultures, consider the conditions under which the organism normally lives. All will usually grow well at 20° C.

You must also consider light as a significant factor. For example, for algae, what sort of light conditions does the particular alga live in? Intense sunlight, filtered light? Consider day length for your organism, if your organism lives in the light. How long is the day when it grows most efficiently? How long is the night? For your slough or river samples, the light is likely to be natural, with some filtering of the light due to the water, silt, and other micro-organisms. You could try growing two samples, one with natural light, one with filtered light.

Note the following from the web page http://cluster3.biosci.utexas.edu/research/utex/media/growth-notes.html

“Of note are cultures which are never placed under bright lights, e.g., the colorless strains of the euglenoids (Astasia, Menoidium, Rhabdomonas and some Euglena strains), the greens (Polytoma, Polytomella and Prototheca); heterotrophs (Ochromonas, Poterioochromonas and Chlorella cultures 1663-1671); and other light-sensitive algae, which are adversely affected by high light intensity. All reds except Porphyridium, the browns, and the macrophytic marine greens grow well in the dimly lighted areas of the culture room or in front of the windows. However, blue-green algae are especially troublesome, because they may bleach out under high light intensity or otherwise achieve little growth in dimly lighted areas. It has been found that most do well on the lighted shelf when the light intensity is diminished by placing a neutral density filter such as a sheet of white bond paper between the cultures and the lights.”

Fungal colonies, according to the web page http://cluster3.biosci.utexas.edu/research/utex/media/growth-notes.html

grow well on vegetable extract media. If you want replicable colours and colony shapes, you are better to use “stock” solutions, which means those made from synthesized chemicals. To prepare vegetable extract media, boil a selection of vegetables (those which you know are willing and able to support fungus) in distilled water for an hour. Strain, saving the water. Check the pH, and adjust to between 5 and 7, using pH paper and sodium hydroxide or hydrochloric acid. (Add it slowly, shake the solution carefully, before rechecking. It is easy to overshoot.) Three kinds of media specifically mentioned on the web page are potato dextrose, and potato carrot, and malt extract. According to the web page, many fungi thrive in slightly acidic conditions. Most need some water, however, condensation in the dishes is a problem. The temperature range under which fungi do best varies considerably but most species seem to grow at 20 to 25 C. Light conditions vary according to the fungus; however, most fungi do not require light and others will die in the presence of light. It would seem that for most fungi, a darker environment is better.

Staining Micro-Organisms for Identification

There are a number of different stains you can use for staining the cells on your slides. Each stain will involve a different method of “fixing” the cells to the slide. Sometimes, the slide will be heat fixed over a flame, sometimes it will be heat fixed over a hotplate, sometimes it will be heat fixed before staining, and sometimes while staining. Some of the stain procedures involve using a variety of stains; others use only one stain. The procedure you use will depend on what you intend to learn from observing the slide.

The stains used in the following recipes are listed immediately below, and the basic ingredients of each stain are listed.

Stains

Congo Red

Purpose: This is a stain that is commonly used to detect bacterial capsules. The stain does not bind to the structure or the capsule but instead stains the glass slide, providing a colored background that outlines the cells.

Composition: Congo red dissolved in distilled water

Physical data: Appearance: brown-red powder. Stable. Incompatible with strong oxidizing agents.

Safety: Congo Red is a possible carcinogen. May cause paternal effects or fetal abnormalities. Harmful if swallowed or inhaled and through skin contact. Irritant.

Personal protection: Safety glasses and gloves. Use only in well-ventilated conditions.

Crystal Violet

Purpose: This is a solution that can be used alone as a simple stain, positive stain or as the primary reagent in the Grams Stain.

Composition:

Solution A:

Crystal Violet

Ethyl Alcohol, 95%

Solution B:

Ammonium Oxalate

Distilled water

Safety procedures: Crystal Violet has very contradictory information on safety. However, the WHMIS site notes Toxic Material Causing Immediate and Serious Toxic Effects. All sites say exposure to skin and eyes is not good, that skin and eyes should be flushed with water for 15 minutes if crystal violet is dropped or splashed on. All sites say one should not take crystal violet internally. Induce vomiting and check with a physician immediately. All sites say the powder can catch fire, if there is sufficient powder in the air and there is an open flame. All sites say that crystal violet is stable, and will last a long time. Ammonium oxalate is an uncontrolled substance, according to the WHMIS web site.

Grams's Iodine Mordant

Purpose: This solution is used as a second reagent (mordant) in the Gram stain. It is valuable because it forms a insoluble chemical complex with the crystal violet, which is bound more strongly to cellular structures. This stain can also be used to detect metachromatic granules, starch and glycogen. The starch and glycogen can be detected by complexes, which retain the solution. This solution can additionally be used to detect starch hydrolysis.

Composition:

Iodine

Potassium Iodine

Distilled water

Instructions: The iodine and potassium iodine should be ground in a mortar. Water is then added in small amounts with further grinding until the solution is dissolved. Additional water should be added, mixed and stored. Remember to always be cautious, this solution should be prepared in a well-ventilated hood.

Malachite Green

Purpose: This solution is a basic dye that is used as the primary stain in the procedure for Schaeffer and Fulton to detect bacterial endospores.

Composition:

Malachite green

Distilled water

Safety: According to the WHMIS website, Malachite green is a “Toxic Material Causing Other Toxic Effects”

Methylene Blue-Loeffler's Alkaline

Purpose: This solution is a solution that can be used alone as a simple stain, positive stain or as the counterstain in the acid fast stain procedure (Ziehl-Nielsen). It can also be used in the staining procedure that detects metachromatic granules (volutin).

Composition:

Solution A:

Methylene blue chloride

Ethyl alcohol, 95%

Solution B: Potassium hydroxide

Distilled water

Safety: Well this is interesting. Research has shown that injecting methylene blue intravenously into animals results in toxicity, and injecting it into the amniotic fluid of pregnant women causes the fetus to become blue and bilirubenic. Why would this be done? At any rate, methylene blue should not be ingested, and should not be injected intravenously, and certainly not while a person is pregnant. There seems to be some carcinogenic effect. The powder is very irritating to the eyes. As with all the chemicals, work with it in a fume hood, wearing gloves. According the WHMIS web site, this is an uncontrolled substance.

Safranin

Purpose: This solution may be used alone as a simple stain, a positive stain or as the counterstain in the Gram Stain procedure. It may also be used in the endospore staining procedures to detect poly-beta-hydroxybutyric acid inclusions.

Composition:

Safranin

Ethyl Alcohol

Distilled water

Safety: According to WHMIS web site, this is an uncontrolled product.

Carbolfuchsin (Ziehl-Nielsen)/Acid Fast Stain

Purpose: This stain solution can be used alone as a simple, positive, or as a primary stain in the acid-fast staining procedure.

Composition:

Basic fuschin

Ethyl Alcohol, 95%

Phenol, heat-melted crystals

Distilled water

The basic fuchsin should first be dissolved in alcohol. The phenol crystals should be heated to 45°C, then transferred to water and dissolved. The basic fuchsin solution should then be mixed with the phenol solution and set aside for several days. The reagent should be filtered for best results.

Calberla's staining solution

Materials:

5ml glycerine

10ml 95% ethanol

15ml distilled water

saturated aqueous basic fuchsin

melted glycerine jelly or glycerol

Method: Mix together a 50:50 solution of fuschin and glycerol. Upon cooling the solution will form a dark pink gel. Store in an airtight jar for future use. Mix the glycerine, ethanol, distilled water and 6 drops of the melted fuschin/glycerol solution. Store the Calberla’s solution in a clear bottle with a dropper lid.

Staining Techniques

There are a number of different staining techniques described below. The first if the gram stain, which can be used to type bacteria as gram negative or gram positive. The second is direct staining for all those stains above which can be used for this very simple procedure. The third is counter staining, which will involve at least two stains, one which will stain either the background slide or a part of the cell, and the other which will stain a different part of the cell.

Gram Stain:

Ingredients: crystal violet, grams iodine, 95% ethanol, safranin

Procedure: Add a loopful of culture to a slide that has a drop of water on it and mix. Air dry and heat fix. Once the slide has been heat fixed the staining procedure is ready to take place. Add Crystal violet for 60 seconds, then rinse off dye briefly with water. Add Grams Iodine for 60 seconds, then rinse thoroughly with water. Add 95% ethanol for 10 15 seconds, then rinse thoroughly with water. Add Safranin for 60 seconds, and rinse off this counterstain briefly with water. Drain and blot.

Congo Red counter stain

Ingredients: congo red, India ink

Procedure: Place a loopful of congo red on a clean glass slide. Mix the loopful of culture in the drop of congo red. Make a thin film of the culture and congo red and air dry on a hot-plate (do not heat fix). Cover the dry smear with India ink. Stain for 60 seconds and drain off ink. Carefully blot the remaining stain with bibulous paper or a paper towel. Do not rinse with water. The smear is now ready to be placed under a microscope and observed for morphological features.

Crystal Violet direct stain

Ingredients: Crystal Violet stain

Procedure: Obtain a loopful of culture and place on a clean slide with a drop of water. Mix thoroughly, then let it air dry. Once dry, heat fix the slide. The slide is now ready to be saturated with a basic dye. Stain the slide with Crystal Violet for 30-60 seconds, then rinse the slide so it is free of unattached dye and blot with a paper towel. The slide is now ready to be observed under the microscope.

Malachite Green counter stain

Ingredients: Malachite Green stain, Safranin stain

Procedure: Endospore Stain: prepare a smear of a culture, air dry and heat fix. place the slide on the rack over the sink. Cover the smear with the malachite green stain and heat until it steams. Continue doing this for at least 5 minutes. (Heat the slide by holding the burner flame above the smear.) Cool the slide for about 1 minute and then rinse the slide with tap water to remove the malachite green stain. Add the counterstain (Safranin) for 60 seconds. The counter stain is a basic stain and it binds to the negative charges on the vegetative cell. The counter stain will not penetrate the endospores. This allows the vegative cells to stain red and the endospores to stain green. Rinse the safranin off of the slide and blot. The slide is now ready to be observed under the microscope.

Methylene Blue-Loeffler's Alkaline cirect stain

Ingredients: Methylene Blue stain

Procedure: Obtain a loopful of culture and place on a clean slide with a drop of water. Mix thoroughly, then let it air dry. Once dry, heat fix the slide. The slide is now ready to be saturated with a basic dye. Stain the slide with Loefflers alkaline methylene blue for 30-60 seconds, then rinse the slide so it is free of unattached dye and pursue by blotting. The slide is now ready to be observed under the microscope.

Acid Fast stain

Ingredients: Carbofuschin stain, acid alcohol, methylene blue

Procedure: prepare an air dried, heat fixed slide with a desired mixed culture. Place the slide over the rack on the sink. Cover the slide with Carbofuschin and heat the stain so that it steams. Do this for at least 5 minutes. (Heat by holding the burner above the slide). If the stain begins to evaporate add fresh stain. Remove the flame if the stain begins to boil. Flood the slide with acid alcohol for 20 seconds then immediately add tap water to stop the decolorizing action. Counterstain with Methylene blue for 60 seconds. Rinse the smear once again and blot dry. The smear is now ready to be observed under the microscope.

Safranin Direct Stain

Ingredients: Safranin stain

Procedure: Obtain a loopful of culture and place on a clean slide with a drop of water. Mix thoroughly, then let it air dry. Once dry, heat fix the slide. The slide is now ready to be saturated with a basic dye. Stain the slide with Safranin for 30-60 seconds, then rinse the slide so it is free of unattached dye and blot. The slide is now ready to be observed under the microscope.

Bibliography

http://physchem.ox.ac.uk/MSDS/C/congo_red.html

http://biosci.calpoly.edu/BioSci/Courses/BACT/BACT221/Unknown/CrystalViolet.htm

http://biosci.calpoly.edu/BioSci/Courses/BACT/BACT221/Unknown/gramsIodMordant.htm

http://biosci.calpoly.edu/BioSci/Courses/BACT/BACT221/Unknown/MalachiteGreen.htm

http://biosci.calpoly.edu/BioSci/Courses/BACT/BACT221/Unknown/Methyleneblue_stain.htm

http://biosci.calpoly.edu/BioSci/Courses/BACT/BACT221/Unknown/safranin.htm

http://biosci.calpoly.edu/BioSci/Courses/BACT/BACT221/Unknown/CarboFuchsin.htm

http://www.dar.csiro.au/Info/airwatch/Appendix_1.htm

http://www.reptox.csst.qc.ca/DetailSimdut.asp?no_produit=156968&nom=Crystal+violet&langue=A

http://www.reptox.csst.qc.ca/DetailSimdut.asp?no_produit=151365&nom=Safranin+O&langue=A

http://www.reptox.csst.qc.ca/DetailSimdut.asp?no_produit=158634&nom=Malachite+green&langue=A

http://www.reptox.csst.qc.ca/DetailSimdut.asp?no_produit=14456&nom=Ammonium+oxalate&langue=A

http://www.reptox.csst.qc.ca/DetailSimdut.asp?no_produit=67&nom=Ferric+chloride&langue=A#7

http://www.reptox.csst.qc.ca/DetailSimdut.asp?no_produit=20115&nom=Methylene+blue&langue=A

http://ntp-server.niehs.nih.gov/htdocs/Chem_Background/ExecSumm/MethyleneBlue/MethBlue_exp_toxeffects.html

Some good sites:

http://warren.dusd.net/~science/communityoflearners/annette/idofbacteria.htm

http://waynesword.palomar.edu/lmexer1.htm

http://www.ebi.calpoly.edu/BioSci/Courses/BACT/BACT221/Unknown/congored.htm