Instructions for Easygel Method

Transcription

1 Instructions for Easygel Method Introduction Please read instructions through completely before beginning! Two simple methods for using Easygel are ) streak (swabbing) plate and ) pour plate. Each is detailed below along with some suggested uses. Microorganisms grow on or in Easygel as colonies or dots in the medium. The colonies may be very small or may spread considerably. They may be colorless, whitish, or various other colors. Bacterial colonies are generally small and solid-looking, while fungus colonies are generally larger and fuzzy-looking. Do not allow students to open the dishes with mold or bacteria in them. If they do, have them wash their hands. You may tape the dishes shut to prevent that occurrence. Plastic petri dishes are intended for one-time use only. Please note: USE ONLY THE PROVIDED PETRI DISHES. Easygel poured in any petri dishes other than those included will not solidify!! Caution: The bottled medium and petri dishes are sterile. Do not contaminate when opening and using the product. Method - Streak Plate (Swabbing) ) Remove petri dishes from their sleeve and lay them on a level surface with the lid side up. ) Remove the cap of an Easygel bottle. Do not touch the rim or inside of bottle, or petri dish. ) Pour all the liquid Easygel into the dish bottom. Replace the lid. Gently swirl to cover the bottom. 4) Wait 45 minutes until the Easygel is solid*. You may now inoculate the dish. Use a wax pencil or magic marker to label the dish. Ideas for use: *Pour dishes a day in advance to save preparation time and give a better quality surface. Clean hands**? Two bottles of Easygel (nutrient or total count), two pretreated petri dishes, and two sterile swabs (Qtips out of a fresh box are generally sterile) are needed. Remove the cap from one of the Easygel bottles and moisten a Q-tip in the liquid Easygel. Wring out the excess liquid against the inside of the bottle as you remove it. Swab a fingertip (unwashed) of the test subject to remove any microorganisms and then insert the swab into the Easygel and twirl it against the inside of the bottle in the liquid to transfer the microbes from the swab to the Easygel. Remove the swab and pour the Easygel-microbe mix into a pretreated petri dish, swirl to cover the bottom and let stand until solid. Wash or disinfect another finger of the test subject and dry it on a clean paper towel. Then repeat the preceding procedure by swabbing the cleaned finger tip and pouring the second petri dish. Put both dishes in a warm place or in an incubator at 5 C. and check at 4, 48 and 7 hours for growth. Colonies of bacteria or molds will develop as spots in/on the solid medium and each colony represents one original cell or colony forming unit (CFU). Count the colonies in each dish and decide if the washing was an effective means of eliminating microbes. ** The first approach is much more effective in illustrating the effects of hand washing than the following commonly used procedure because pressing a very contaminated fingertip on a nutrient gel will often result in so many bacteria growing that an almost imperceptible film results and interpreters will think that nothing grew, while from the clean finger will result in a number of large colonies that are much more obvious and the inexperienced person will think that more growth was present than on the dirty finger. Divide the Easygel dish into halves by turning the dish over and drawing a line across the bottom with a wax pencil or magic marker. Remove the lid and touch several fingers to the surface on one half. Wash your hands thoroughly with soap and water, dry on a clean paper towel, and touch the same fingers (washed) to the other half. Label and incubate. Check at 4, 48, and 7 hours for growth. Did you get less growth after washing? Why or why not?) Clean door knob or table? Use a sterile swab which has been moistened by dipping in the Easygel** to wipe (rotating the swab so that the entire swab makes contact) a door knob/handle or table/counter top. Remove the lid of a dish and starting on one side of the dish gently wipe the swab against the surface of the gel, rotating the swab, while moving in a zig zag pattern. Wiping too hard will tear the gel. Put the lid back on the dish and incubate. Check at 4, 48 and 7 hours for growth. Insect Inoculation: Using a solidified dish of Easygel, place a live insect on the gelled medium, and replace the lid. Remove the insect after it has moved around the surface and place the lid back on the dish and incubate for 48 hours. Other inoculum ideas: Gently touch to a dish a: dirty sock, old toothbrush, dust, pencil, etc. Use imagination. **If the dishes are poured a day in advance, use the remaining residue in the Easygel bottle to moisten the swab.

2 Method - Pour Plate ) Remove the cap of an Easygel bottle and add the desired amount of sample. Swirl gently to distribute the sample. ) Lift the lid of a pre-treated petri dish and pour the Easygel/sample mixture into the dish bottom. Replace the lid and gently swirl until the bottom is covered. ) Allow 45 minutes for solidification. Invert and incubate as desired or allow the dishes to remain un-inverted for incubation and eliminate the wait for solidification. Idea for Use: Water Testing: Using the above pour plate instructions, use a sterile pipet to add milliliter (or alternatively 0. ml-- approx. drops) of water from a puddle, pond, lake, stream, etc. to the liquid Easygel as your sample. Incubate at room temperature for 48 hours. Count the colonies and determine how many there are per ml. Disposal Disposal of the used petri dishes may be accomplished through autoclaving or heating in an oven-proof bag for 45 minutes at 00 degrees. Or just add a teaspoon of household bleach to cover the surface of the medium in the dish and allow to stand for 0 minutes which will kill the microbes in the dish, or make a 0% solution of bleach in a bucket and drop the dishes in the disinfectant solution (later pouring off the solution and disposing of the dishes in the garbage). Be sure that fresh bleach is used for each batch of dishes as the chlorine will dissipate when the bleach stands in the open. The bleach should smell of chlorine. For more information contact: Micrology Laboratories, LLC. 0 Eisenhower Drive S. Goshen, IN USA Phone: or Fax: micrologylabs@juno.com Easygel is a registered trademark of Micrology Laboratories, LLC., Goshen, IN 4656 USA

3 Easygel Method Procedure USE ONLY THE PROVIDED SPECIALLY COATED PETRI DISHES Pour Plate Instructions: Option! " #$! % & ' ( $) % * ' %! &+, $ - Pour Plate Instructions: Option DO NOT., ) % " ' ( $) % * ' %! &+ Streak Plate Instructions: %/ ) %. % ) 0 - %! $ - Trouble Shooting * $! %! " 4 % ) )%% % 56 )66*)(777-"8-9&",,8&-,""-"",/!(,8&-,""-""8+

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5 Selective For: Description: Composition: Procedure: E. coli and other coliforms Coliscan is a clear medium containing two chromogenic substrates. One is cleaved by the enzyme galactosidase (produced almost exclusively by coliforms) to produce an insoluble pink compound. This causes coliform colonies to be pink/red. The second is cleaved by the enzyme glucuronidase (produced almost exclusively by E. coli) to produce an insoluble teal compound. Using the two compounds allows easy visual distinction between general coliforms (pink/red colonies) and E. coli (pink + teal = blue/purple colonies). Contains nutrients, inhibitors and chromogenic substrate mix. ph 7. ± 0. Use standard methodology for product being tested. Incubation time and temperature; 4-48 hrs at 5ºC. It is possible to take accurate readings at 4 hours incubation time. Colors will intensify and colony size will be larger at 6-48 hours. Interpretation: E. coli* colonies will have a dark blue/purple overall colony color. Other coliforms (Enterobacter, Citrobacter and Klebsiella spp. & others) are pink to red in color. Proteus and Salmonella will generally grow as white colonies. Gram-positive organisms are generally inhibited. An occasional teal colony is indicative of a non-coliform which has the ability to produce glucuronidase but not galactosidase. A few strains of Salmonella and Shigella have this capability. Storage: Shelf Life: Freeze year "#$ % "%!! Phone: 888-EASYGEL Fax: Web: info@micrologylabs.com

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9 Selective For: Sorbitol positive E. coli and other coliforms, and Sorbitol negative, glucuronidase negative E. coli. Description: Composition: Procedure: A pink medium for the identification and differentiation of beta glucuronidase positive, sorbitol positive strains of E. coli and glucuronidase negative, sorbitol negative strains of E. coli. Other coliforms which are sorbitol positive will also be identified. Contains nutrients, a chromogenic enzyme substrate mix, the sugar sorbitol, and a ph indicator. ph 7.8 ± 0. Use standard methodology for product being tested. Incubation time and temperature; 0-0 hrs at 5ºC. Crowded colonies (CFUs) closer than -4 mm on the dish may result in difficult to interpret background zones (color). Interpretation: Standard glucuronidase positive, sorbitol positive E. coli CFUs are dark blue/purple surrounded by a yellow zone. Other coliforms (galactosidase positive, sorbitol positive) are red surrounded by a yellow zone. Glucuronidase negative, sorbitol negative E. coli (includes 057:H7) CFUs are red without a surrounding zone. It is common that both the standard E. coli and the sorbitol negative E. coli colonies are larger than the colonies of the other coliform types. Storage: Shelf Life: Freeze year #$% & #& "' Micrology Laboratories, LLC. PO Box 40 Goshen, IN USA Phone: 888-EASYGEL Fax: Web: info@micrologylabs.com

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11 Selective For: Description: Composition: Procedure: E. coli, other coliforms, Aeromonas spp., and some Salmonella spp. ECA Check is a clear medium containing a chromogenic substrate mixture. Enzymes specific to each of the four bacterial groups react with their specific chromogenic substrates to produce the colors that differentiate each bacterial type. Contains nutrients, inhibitors and chromogenic substrate mix. ph 7. ± 0. Use standard methodology for product being tested. Incubation time and temperature; 4-48 hrs at 5ºC. It is possible to take accurate readings at 4 hours incubation time. Colors will intensify and colony size will be larger at 6-48 hours. Interpretation: E. coli colonies will have a dark blue/indigo overall colony color. Non- fecal coliforms (Enterobacter, Citrobacter and Klebsiella spp. & others) are lighter blue/grey to purplish in color. Salmonella spp. appear as green/teal colonies. Aeromonas spp. are pink to very light pink. Gram-positive organisms are generally inhibited. Storage: Shelf Life: Freeze year %&' ( %( $! Phone: 888-EASYGEL Fax: Web: info@micrologylabs.com

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13 Selective For: Description: Composition: Procedure: E. coli, other coliforms, Aeromonas spp., and some Salmonella spp. ECA Check Plus is a clear medium containing a chromogenic substrate mixture. Enzymes specific to each of the four bacterial groups react with their specific chromogenic substrates to produce the colors that differentiate each bacterial type. In addition, the glucuronidase cleaves the fluorescent entitywhich results in a bright blue fluorescence from E. coli colonies lighted by longwave UV light. Contains nutrients, inhibitors and chromogenic substrate mix. ph 7. ± 0. Use standard methodology for product being tested. Incubation time and temperature; 4-48 hrs at 5ºC. It is possible to take accurate readings at 4 hours incubation time. Colors will intensify and colony size will be larger at 6-48 hours. The fluorescence of E. coli can generally be detected by -5 hr. incubation. Fluorescence may diffuse throughout the plate after 4 hrs making quantification by fluorescence impossible after that initial time period. Interpretation: E. coli* colonies will have a dark blue/purple overall colony color and fluoresce a bright blue under long wave UV light. Non-fecal coliforms (Enterobacter,Citrobacter, Klebsiella spp. and others) do not fluoresce and are light blue/grey to purplish in color. Salmonella spp. do not fluoresce and appear as green/teal colonies. Aeromonas spp. do not fluoresce and are pink to very light pink. Gram-positive organisms are generally inhibited. Storage: Shelf Life: Freeze year +), " ( +( Phone: 888-EASYGEL Web: info@micrologylabs.com

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16 Swab Method Procedures for Easygel Introduction There are two swabbing methods with Easygel. The first is to use the Easygel to moisten a swab and then swab a surface. The second is to pour the bottle of Easygel into the dish, allow it to solidify, and then swab a surface and streak the swab across the surface of the solidified dish. The first method is better for inexperienced persons. Method (Liquid moisten, swab and pour) ) Remove a swab from sterile package. Do not touch swab tip or touch it against anything. ) Moisten the swab if necessary using either the Easygel liquid, sterile water, or sterile buffer. Wring excess liquid against inside of bottle. (Step is optional) ) Swab the surface(s) you wish to monitor, rotating as you swab. It is recommended that you test a surface by swabbing square inches. 4) After swabbing, immerse the swab in the Easygel and twirl back and forth while pressing against the inside of the bottle. Break or cut the swab end (above the tip) into the bottle of Easygel. Swirl the bottle to mix. 5) Open a pretreated petri dish and pour the contents of the bottle (minus the swab) into the dish. Replace the lid and gently swirl to cover the bottom. 6) Allow 45 minutes to solidify. After the liquid is solidified you may want to tape the lid on, and then incubate. Incubate either in an incubator or in a warm place. DO NOT place in direct sunlight or over a direct heat source, radiator, furnace duct etc. You may place dishes near one of these sources or in a warm spot in the kitchen. Allow 4-48 hours for growth to begin. Once growth begins you can incubate another 4 hours for complete growth to take place. 7) After 7 hours, read and record the results. To calculate: # of colonies # of square inches = # of colonies per square inch Example: 80 colonies square inches = 90 colonies per square inch Method (Pour, solidify and swab) (Prepare dishes one or more days in advance to save preparation time and give a better quality surface.) ) Remove petri dishes from their sleeve and lay them on a level surface with the lid side up. ) Remove the cap of an Easygel bottle. Do not touch the rim or inside of bottle, or petri dish. ) Pour the liquid Easygel into the dish bottom. Replace the lid. Gently swirl to cover the bottom. 4) Allow 45 minutes for the Easygel to solidify. You may now inoculate the dish. Use a wax pencil or magic marker to label the dish. 5) Swab the surface(s) you wish to monitor, rotating as you swab. It is recommended that you test a surface by swabbing square inches. 6) Gently streak the swab across the surface of the gel in a long zig-zag pattern back and forth. Replace the lid and incubate. (You may want to tape the lid down.) Incubate either in an incubator or in a warm place. DO NOT place in direct sunlight or over a direct heat source, radiator, furnace duct etc. You may place dishes near one of these sources or in a warm spot in the kitchen. Allow 4-48 hours for growth to begin. Once growth begins you can incubate another 4 hours for complete growth to take place. 7) After 7 hours, read and record the results. To calculate: # of colonies # of square inches = # of colonies per square inch Example: 80 colonies square inches = 90 colonies per square inch Easygel is a registered trademark of Micrology Laboratories, LLC Micrology Laboratories, LLC. Phone: or Fax: