Lab 1: The Dilution Plate Technique for Isolation of Soil Fungi

Team: Collin Campbell and Chan Uppuluri

BIO 4942 Mycology

October 24, 2010

Introduction

Isolating fungi from different substrates is a key step in analyzing environmental constituents and

comparing populations among different communities. In order to compare different communities, one

has to make sure that no other extraneous factors are influencing the results. Like many comparative

studies, scientists can cut out extraneous factors by standardizing the experimental conditions. Of the

various methods of isolating fungi, dilution plate technique was utilized in this experiment to

standardize the community samples for our purpose of comparing our different communities.

Materials and methods

Two soil samples each of at least 5.00 grams or more were obtained from two different communities.

From each community 5.00 grams of soil were saved in two glass containers to dry (to later compare

moisture levels of the different communities.) The other two samples from the two communities were

mixed in a dilution series. The dilution series consisted of diluting 5.00 grams of soil in 100.00 mL of

sterile water. The mixture was shaken up to homogenize the constituents of the bottle. Then while the

mixture is still “in motion” before settling, 1.0 mL of the first dilution is then added to another bottle of

49.0 mL of sterile water, creating a dilution of 1:1,000. The second bottle was shaken up again to

homogenize the mixture. While that mixture was in motion, another 1.0 mL of mixture was transferred

to a test tube of 9.0 mL of sterile water, creating a 1:10,000 dilution. To plate, 1.0 mL of dilution 3 was

transferred to petri plates containing glucose peptone rose Bengal agar that was treated with

streptomycin and penicillin, and spread evenly across the plate in circular motions with a glass “hockey

stick” shaped rod. The plates were stored in a Ziplock bag and incubated for 7 days. Of the plates

showing viable fungal growth, 20 colonies were transferred to separate Petri dishes of potato dextrose

agar for isolation by transferring each species’ fungal hyphal fragments to a different plate. After

another 7 days, these new plates were once again analyzed for purity of isolates. Pure fungal isolates
were transferred to tubes of various media by the same method of fragmentation of fungal hyphae.

Impure isolates were transferred to more PDA plates by the same methods for further isolation.

Results

Although there were 61 original colonies from both communities, after plating differing separate

colonies, there was a total of 34 cultured fungal isolates across the two communities (Table 1.) The two

communities from the Alley (community A) and the Pine tree (community B) soil had a 0.098 coefficient

of similarity. Only three species of fungi were present in both communities from the 30 colonies in

community A and the 31 colonies in community B (Table 2.) The relative densities of species were rather

even save for the extra prevalence of species M and L (Table 2.) Both communities had a rather even

number of isolates despite the varying water content. Community A soil was 23.5% water whereas

Community B soil was only 8.60% water content (Table 3.)

Discussion

The two communities had rather prevalent species present although the number of different types of

species could be greatly reduced by identifying similar and different colonies. Across the two

communities, the populations had very little similarity in their species content – almost all of the

exhibited colonies were different in both communities. Each species seemed to have equal opportunity

in each community except for species M and species L that seemed to over grow and exhibit multiple

colonies despite isolating separate colonies. This indicates a good diversity among the communities and

a noticeable difference of the different types of fungi that will be more prevalent in an area of pine tree

soil versus an area of alley soil. We may be able to expect repeatable results of such a difference

between two dissimilar communities. Finally, both communities exhibited a similar number of fungal

colonies (30 versus 31) despite the largely varying water content in each community. This may indicate

that these fungal spores can survive despite being in such different environments, and that fungal
spores may be able to resist harsh/dry conditions. Whether or not these spores would have successfully

germinated is an issue that requires further observation in a more natural/less synthetic system.