Lab #2, Soil Bacteria and FungiForests Through Time and Space Winter 05Objectives• Estimate the number of colony-forming units (CFUs) of bacteria and actinomycetes per gram soil.• Become familiar with serial dilutions and spread plating.• Calculate the number of CFUs per gram soil using spread plate counts• Become familiar with the macroscopic and microscopic features of soil bacteria and fungiWhat to Bring to Lab• Soil sample (app 50 g)BackgroundThere are a number of different methods for determining the number of microbial colony-forming units(CFUs) in soil. The results depend on diluting the CFUs to a concentration where the growth of one colonydoes not inhibit neighboring colonies. Bacteria are the most numerous culturable organisms in soils (virusesare more numerous, but difficult to culture—you need a suitable host). The main species are Arthrobacter,Bacillus, Pseudomonas, and Streptomyces. Arthrobacter, and Streptomyces are actinomycetes whichproduce cells that resemble fungi. The results you will get depend on the media used. Media for isolatingbacteria is usually nutrient poor to inhibit the growth of fungi, while that used for fungi often has antibioticsand is acidified to inhibit bacterial growth.There are a number of potential problems with evaluating the results of these assays in terms of what theymean in the soil. Nevertheless, these techniques are still commonly used to compare soils. Think carefullyabout the assumptions that lead to the numbers you get. You will be asked to speculate on this in yourreport.MethodsSerial Dilution (week 1)Materials—1 sterile 95 ml water blank, 1 sterile 99 ml water blank, 5 sterile 9 ml water blanks, 6 sterile 1 mlpipettes1. Label five 9 ml water blanks with 10-4, 10-5, 10-6, 10-7,and 10-8.2. Weigh out 10.0 g of moist soil (record the exact weight) and put into a 95 ml water blank. Capand shake well (about 10 minutes).3. Using a sterile pipette, transfer 1.0 ml of the original suspension into the 99 ml water blank(creating the 10-3 dilution). Cap and shake well (about 5 minutes).4. Using a new sterile pipette, transfer 1.0 ml of the above suspension to the first 9 ml water blank(10-4). Cap and shake or vortex well (3–5 minutes).5. Repeat step 8 using the 10-4 dilution as the source and transferring 1.0 ml into the 10-5 blank.Repeat for all subsequent dilutions (Fig 1).Inoculation of spread plates (week 1)Materials—six plates each of tryptic-soya agar and Martin’s Rose Bengal agar, alcohol lamp, glass hockeystick, jar of 95% EtOH to dip hockey stick, 3 sterile 0.1 ml pipette tips and pipettor, 2 plastic bags to holdplates (Fig 1).Lab #2 Soil Bact & Fungi.doc Page 2 of 36. Label two plates of each media on thebottoms with the media type, dilution,and your names for the three mostdilute solutions (10-6, 10-7,and 10-8).7. Beginning with the most dilute soilsuspension, shake and, using a sterilepipette, transfer 0.1 ml to each the fourplates for that dilution (1 of eachmedium) (Fig 1).8. Dip the hockey stick into the EtOH tosterilize it and lightly flame it toremove the EtOH. Allow to coolbriefly.9. Briefly touch the hockey stick to theagar to cool in an inoculated plate awayfrom the inoculum (don’t want to cookthe inoculum). Spread the inoculum bymoving the hockey stick in an arc whilerotating the plate. Continue until all thewater has been absorbed into the agar.Repeat steps 16 and 17 for the otherplate for this dilution.10. Repeat steps 15–17 for the other twolowest dilutions.11. Put the plates in a plastic bag (all onemedium together) and incubate theplates inverted for one week for thebacteria. Paul will check on the platesand put them in the cooler whenthey’ve grown sufficientlyObservation and Enumeration (week 3)Materials—inoculated plates from last week , counter, marker, inoculating loop, ethanol, microscope slides,alcohol lamp, compound scope, immersion oil.In order to get an accurate count, you need between 20–200 colonies per plate. Plates that fall outside of thisrange won’t give accurate estimates. Observe not only the quantity, but also the qualitative aspects of thecolonies. Bacteria generally appear smooth and creamy, or mucoid. They can be discrete round colonies,spreading or feathery. Actinomycetes typically have a white, gray, or black powdery surface, and are firmand leathery. Actinomycete colonies will break under pressure and resist movement when scraped with aneedle while bacteria will smear. A clear zone indicative of antibiotic production often surroundsactinomycetes.12. Count the appropriate dilution plates for each medium. Record the numbers of bacteria &actinomycetes on the tryptic-soya agar and the number of fungi and actinomycetes on theMartin’s Rose Bengal agar.13. Observe representatives of the various microorganisms through the compound microscopes asoutlined below.Figure 1. Serial dilution and plating techniqueLab #2 Soil Bact & Fungi.doc Page 3 of 314. Transfer a small drop of tap water for a slide with an inoculating loop. Flame the loop andremove a small amount of culture. Mix the bacteria in the drop of water, spreading it over an areaabout the size of a dime.15. Cover with a cover slip and examine first at 400x (40x objective)16. Examine the slide using oil and the immersion objective. Describe and sketch what you see.17. To examine fungi, remove a small bit of mycelium (can include a little agar too) from a fungalcolony using a dissecting needle as follows:a. Flame the needle, quench, and flame off EtOH.b. Slowly open petri dish just enough to get needle into colony, open the dish away fromyou, or to the side.c. Cut a small bit of mycelium and transfer it to a slide.d. Add a half drop of KOH and a half drop of phloxine, mix with mycelium, put coverslipon and gently squish. If you label one end of your slide, you can do two or threespecimens per slide.e. Examine the slide using the 40X objective, looking for clamp connections (ask if youhave any doubts). Most of the white-rot basidiomycetes, as well as other basidiomycetes,have clamp connections between adjacent “cells” in the mycelium. Record all yourobservations with sketches.18. Repeat the process in 17 for any suspected Actinomycetes. Sketch and describe what you see.CalculationsYou have the data to estimate the number of CFUs per gram dry soil for bacteria/actinomycetes and fungifrom the spread plates.Spread Plate Calculations1. Calculate the grams of soil per ml in your original solution