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Name: Matthew He
MIMM 212 – Laboratory in Microbiology
TA: Marieme Dembele
Due Date: October 26th, 2017

MIMM 212 Lab Report 1:

Amount and Diversity of Bacteria in Colour Lake Sediment

Abstract:

Soil contains an entire ecosystem of bacteria and could very well hold the key to the next mass market

antibiotic, and a McGill researcher has collected soil samples from the Arctic region to be studied for this

reason. However, the information regarding these soil samples do no got beyond their names and

where they were collected. In the following work, we used soil characterization, dilution and plating,

colony counting, and picking and patching techniques to obtain a deeper understanding of both the soil

and its interactions with various media. Our results show that the Colour Lake Sediment soil sample is a

sandy loam type soil with a pH of 5, and that its’ bacteria grows better on Blood Agar Plate (BAP) media

than on G. LB media and TSB G media at a temperature of 25oC, showing the colonies to be mesophilic

and highly competitive. This information will be used to design the best antibiotic screening test for this

specific sample.

Introduction:

A McGill researcher brought down soil samples collected around the McGill Arctic Research Station in

Nunavut to be tested and screened for antibiotic activities. Our group was given a soil sample collected

near Colour Lake, aptly named Colour Lake Sediment (CLS). The majority of antibiotics used today have

been isolated from soil samples (Schmieder & Edwards 73); the soil environment places the bacteria

living in it under very high competitive stress, and as a result, these soil bacteria have evolved various
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molecules and mechanisms that interact with its surroundings, some of which produce antibiotics that

inhibit the growth of other microorganisms (Hernandez et. al 19).

The hypothesis of this experiment is that the CLS soil samples are diverse and will contain billions of cells

per gram. The rationale for the soil samples’ diversity is that the soil environment places high

competitive stress of the soil bacteria (Hernandez et. al 19), and thus the soil bacteria will diversify in

structure and environmental interaction mechanisms. The rationale for the billions of cells per gram of

soil comes from research by Schloss & Handelsman, in which they state, “one gram of soil can contain

billions of individual cells from tens of thousands of bacterial species” (2006). The main objective of this

experiment is to test the hypothesis by characterizing the soil sample in both qualitative and

quantitative methods. The end result is to gain a better understanding of the unknown soil sample.

The methods employed to gain a better understanding were to characterize the soil sample, perform a

serial dilution series and spread plate, calculate the colony forming units after incubation, and pick and

patch colonies of interest after sufficient growth onto new plates for isolation. The characterization of

soil is significant as the texture of soil affects many other chemical, physical, and biological soil processes

(Thien 1), all of which affect the types of microorganisms capable of living in it and the antibiotic

properties that they may have. Thiens’ “guide to [soil] texture by feel” allows for the simplest

characterization of the unknown soil sample. This information, along with results from a pH test, allows

for a full set of characterizations across all soil samples that can be cross referenced to examine trends

relating to them and antibiotic activity. A serial dilution is performed to make samples of known

concentration that contain less and less bacteria in each while retaining the high diversity. This is done

so that upon plating and incubation, we may observe and count the number of individual colonies. By

knowing the number of individual colonies, the concentration, and the amount of sample plated, we can

calculate the number of colony forming units per gram of soil on that specific medium. This allows for

recognizing what the optimal environment for the unknown mixture of bacteria in the soil sample may
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be. As soil bacteria is incredibly diverse, the plates with diluted samples would show colonies of all

different types of morphologies. Picking and patching allows for isolating a set of bacteria that are

largely different from each other onto a single plate, where they may grow without interference from

other bacteria.

Methods:

Characterization of Soil Sample

We were given a soil sample and the first task was to characterize it beyond its name. To do so, the soil

“guide to texture by feel” [A], from S.J. Thien, 1979, was used. This is a flow chart of steps, each ending

with a yes or no question that leads to either another step or a resulting soil type. A dime sized sample

of the soil was placed inside the palm of a gloved hand, and water droplets were added until the soil

resembled moist putty, kneadable and capable of holding a ball shape when squeezed. Then, the soil

sample was shaped by the thumb and the forefinger into a ribbon until it collapsed, and the length of

the ribbon right before breaking was used to determine the following texture characterization steps,

which asked for a small pinch of soil to be excessively wet with water and rubbed on the palm with a

forefinger. The texture from this test was used to determine which type of soil it is. Following the

texture characterization, the stock solution for the serial dilution was made (which will be discussed in

length next), and a pH test was done by adding drops of the stock solution to a pH indicator paper. The

color of the pH paper was then compared to the guide that comes with the paper, and an approximate

pH range was found. Through these two tests, the soil sample was characterized.

Serial Dilution and Spread Plating

The serial dilution requires a balance, one large conical tube, four 1.5mL microcentrifuge tubes,

micropipettes ranging from 100uL to 5mL and their respective tips, test tube and centrifuge tube racks,

distilled water (dH2O) and a vortex mixer. The serial plating requires additionally 5 plates of a chosen
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media type and a t-spreader. To begin, 1 gram of the soil sample was measured and added to a large

conical tube with 9 milliliters of dH2O to make the stock solution. The large conical tube was vortexed

for 15 minutes on various angles to dissolve soil in the solution. The four 1.5mL microcentrifuge tubes

were filled each with 900uL of dH2O and labeled 1, 2, 3, and 4 to represent their dilution number. 100uL

of the stock solution was then pipetted into the microcentrifuge tube labeled 1, which was then capped

and vortexed for 1 minute to create dilution 1. 100uL of dilution 1 was then pipetted into the

microcentrifuge tube labeled 2, which was then capped and vortexed to create dilution 2. This series

was repeated twice more to form dilutions 3 and 4. Then, a medium was chosen for spread plating, and

5 plates of the medium were labeled. Starting from the lowest dilution, dilution 4, the microcentrifuge

tube was vortexed and 100uL was pipetted to its corresponding plate. The t-shaped spreader was used

to evenly distribute the solution over the surface of the plate before taping the lid to the plate. This was

repeated for the remaining dilutions, in the order of ascending concentration (dilution 3  dilution 2 

dilution 1  stock). The finished plates were inoculated upside down at the chosen temperature, and

the remaining solution was discarded as hazardous waste. The number of colony forming units (CFUs)

were counted after 48 hours of incubation, and then once more after 5 days of incubation.

Colony Forming Units

After allowing the spread plates to incubate for 48 hours, observations were made on the amount of

visible, individual colonies on each of the five plates. If there were more than 300 visible colonies, it was

deemed to be too numerous to count – TNTC. If there were less than 30 visible colonies, it was deemed

to be too few to count – TFTC. Anything in between 30 and 300 was noted down as a number. The

observations were recorded, and were made and recorded again after a total period of 5 days.

Calculations were then made to determine the colony forming units per gram of soil by choosing a

numerical observation, multiplying it by the total dilution factor, and dividing by the amount of solution

plated in mL. The CFU/g of the soil sample on each type of media was recorded.
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Picking and Patching

Two clean plates of a chosen media were labeled with appropriate information similar to that in the

serial dilutions step, and one was labeled “working plate” and the other “backup plate”. Identical grids

were drawn on the back of each plate, and a mark was made to indicate the top of the grid. The serial

dilutions plates that had individual, unmixed colonies were then opened, and using a toothpick, a colony

was picked off from the dilution plate and placed by a zig-zag motion onto the first grid on the working

plate surface, and then the same grid on the back up plate. This was repeated a total of 18 times,

replacing toothpicks each time, until all grids had bacteria occupying it. Observations of the morphology

of each picked colony was recorded, and the plates were taped up and incubated at 25oC, upside down.

Results:

Characterization of Soil Sample

The Colour Lake Sediment sample remained in a ball when squeezed, formed a weak ribbon, and felt

gritty. The soil type of CLS was determined to be sandy loam. The pH was found to be 5.

Colony Forming Units

The media used for plating were G. LB, BAP, and TSB-G 10%. Lab instruction prescribed G. LB media, and

it has industry standard status in cultivating E. coli (Miller, 1972), which reflected applicability to familiar

bacteria. BAP media was chosen for its’ high availability and growth facilitation for all types of bacteria

(Hernandez et. al 26), and TSB-G 10% media was chosen at random. The results after counting the

colony forming units and calculating the colony forming units per gram of soil at different conditions and

medias are as follows. After 2 days of 25oC incubation on G. LB media, the CLS sample showed to have

4.5 x 105 CFU/g. After 7 days of 25oC incubation on G. LB media, the CLS sample showed to have 6.9 x

105 CFU/g. After 5 days of 25oC incubation on BAP media, the CLS sample showed to have 7.1 x 106
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CFU/g. Lastly, after 7 days of 4oC incubation on TSB-G 10% media, the CLS sample showed to have 3.6 x

106 CFU/g. The data taken may be viewed in Table 1.

Table 1: Color Lake Sediment Serial Dilution 100uL Plating CFU Summary
Sample from G. LB 2 Days 25oC G. LB 7 Days 25oC BAP 5 Days 25oC TSB-G 10% 7 Days 4oC
Stock TNTC TNTC TNTC TNTC
Dilution 1 TNTC TNTC TNTC TNTC
Dilution 2 45 69 TNTC TNTC
Dilution 3 TFTC TFTC 71 36
Dilution 4 TFTC TFTC TFTC TFTC

Table 2: Color Lake Sediment Serial Dilution Results Summary

Sample from G. LB 2 Days 25oC G. LB 7 Days 25oC BAP 5 Days 25oC TSB-G 10% 7 Days 4oC
5 5 6
CFU/g soil 4.5 x 10 6.9 x 10 7.1 x 10 3.6 x 106

Below are photos from the Color Lake Sediment serial dilutions used for CFU counting. The diversity of

colonies is particularly visible in the BAP media sample. This may be attributed to the increased contrast

on the red media.

G. LB 2 Days 25oC – Dil 2 G. LB 7 Days 25oC – Dil 2 BAP 5 Days 25oC – Dil 3

Note: No pictures were taken of the TSB-G 10% 7 Days 4oC samples.

Picking and Patching

The media used for picking and patching were BAP Cyclo and G. LB media, and the chosen temperature

of incubation was 25oC. 18 grids were drawn for the BAP Cyclo plate; grids 1 to 11 were picked from BAP

Dilution 3, grids 12 to 14 were picked from BAP Dilution 2, and grids 15 to 18 were picked from BAP
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Dilution 1. 24 grids were drawn for the G. LB plate, and all samples were picked from G. LB Dilution 2.

Below are photos from the most recent Color Lake Sediment pick and patching plates, taken after two

days of growth at 25o C.

G. LB Pick and Patch, Post 2 Days at 25oC BAP Pick and Patch, Post 2 Days at 25oC

Table 3: Morphology Description of Picked Colonies

Grid Number BAP 5 Days 25oC G. LB 7 Days 25oC
1 White, round, glistening Wrinkly, irregular, pale, yellow, smooth, glistening
Yellow, white centre, umbonate, smooth, glistening,
2 Orange, round, dull
circular
3 White and red, irregular Pink beige, irregular, convex
4 White, round, glistening Smooth, glistening, beige, translucent, circular
5 Orange, round, dull Opaque, yellow-orange, circular
Did not spread. Raised squiggly lines in colony, white
6 Pink, round, glistening
beige, dry powdery
7 White, round, glistening Yellow, circular, smooth, glistening
8 Pink, round, glistening Dry, wrinkly, irregular, beige
9 Pink, round, glistening Beige, smooth, glistening, circular
10 White, round, glistening Punctiform, yellow, entire, smooth, glistening, raised
11 Yellow-orange, round, glistening White beige, cloudy translucent, irregular
12 Pink, large, glistening Smooth glistening, orange-beige, raised circular
13 Pink-orange, large Small, yellow, circular, smooth, glistening
14 Large, white, cloudy, irregular Bright yellow, circular, smooth, glistening
15 Black and dark purple Beige, circular, raised, smooth, glistening
16 White, round, glistening Beige, brown, circular, smooth, glistening
17 Pink, glistening Yellow, circular, smooth, glistening
18 Orange, glistening Beige, circular, smooth, glistening
19 Wrinkly, white, will not spread
20 Small, yellow, punctiform, smooth, glistening
21 Beige-brown, circular, smooth, glistening
22 Yellow-beige, convex, circular, smooth, glistening
23 Yellow, small, circular, smooth, glistening
24 Beige-brown, circular, smooth, glistening
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Discussion:

The identification of the Colour Lake Sediment as sandy loam gives further insight into the composition

of the sample. According to R.B. Brown, sandy loam is made of approximately 60 percent sand, 10

percent clay and 30 percent silt particles (4). Sandy soils generally are low in organic matter, fertility,

moisture, and nutrients (Brown, 4), giving the bacteria situated inside it a very tough environment to

flourish in. This may affect the secondary metabolites the bacteria produce in response to the

environment stimuli, and may be reflected in the antibiotics produced by the sample. The pH of the

sample shows that these bacteria may be well adapted to a slightly more acidic environment, and may

prove interesting to test against pathogens that flourish more in acidic environments.

The results of the colony forming units show that BAP media and 25oC incubation is the more optimal

choice for the CLS sample because in the same amount of time, it formed over ten times the colonies

than that of G. LB in the same temperature, and in a less amount of time, it formed nearly double the

colonies than that of TSB-G 10% at 4oC. These results influenced the choice of BAP media for the picking

and patching portion, as the goal is to have a set of diverse colonies. BAP media has also been shown to

be excellent for growth of all types of ESKAPE pathogens (Hernandez et. al 26), and it is reflected in the

numerical growth of the bacterial colonies in our experiment. The colonies that were patched onto the

BAP working and backup plates all came from BAP serial dilution plates as to preserve constant

conditions and encourage good growth. The other media used for picking and patching was G. LB media

and it was incubated at 25oC too. G. LB was found to have the second highest CFU/ g, and this finding

influenced it choice as the second media.

An unexpected colony grew on the dilution-2 plate of the BAP serial dilutions; while all other colonies

were white, cream, orange, or yellow in color, that particular colony grew as a black center with and

extending, uneven ring of dark purple around it. This unusual colony was picked and patched onto
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position 15 of the patch plates. According to a genome sequencing project done by D. Bryant and N.

Frigaard on phototrophs, purple bacteria are proteobacteria that are capable of photosynthesis, and can

oxidize sulfide, thiosulfate and H2 while being capable of fixing carbon by the reductive pentose-

phosphate cycle (D. Bryant and N. Frigaard, 488).

The serial dilution plates showed that despite the high amount of growth on the BAP plate, the diversity

of colony morphologies was less than that of the GLB plates. The BAP serial dilution plates produced

only five types of distinctively different morphologies, with their differences mainly being their colors

and not much difference in texture, sheen, or edge shape. The GLB plates, with a magnitude less of

colony growth, contained colonies of various morphologies mainly differing in texture. This may mean

that BAP media is well suited for a small selection of bacteria and not for the rest, while the GLB media

supplies a larger selection of bacteria, but less well for each. This can also help explain why a larger

number of colonies form on BAP media: since it provides more selectively and to a smaller amount of

bacterial species, there is less interspecies competition, as opposed to the GLB media where the large

amount of species on it means a lot more competition.

Sources of error in this experiment would be mainly attributed to human error. Although aseptic

technique was practiced, the long-period-open-lid nature of the plating, picking and patching portion of

this lab may have allowed dust and other contaminants to settle on the surface of the media, causing

undesired colonies to grow. Another source of error may come from how well the serial dilution plates

were spread. A t-spreader cannot ensure perfect distribution of the 100uL of diluted solution and could

result in areas of higher concentration of bacteria on one plate, meaning less competition in the other

areas of the same plate. This could cause otherwise unfit colonies to grow, and similarly, the raised

competition on one side could kill off more colonies that would have otherwise survived. These errors

can only be reduced, not eliminated completely. The best way to avoid contamination would be to work
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in a highly aseptic environment, and the best way to ensure even spreading would be to use plating

beads rather than plastic spreaders.

Overall, the experiments went smoothly. 25oC was found to be better suited for incubating bacteria than

4oC, and this will prove beneficial for antibiotic activity screening as the ESKAPE pathogens are

mesophilic (ALS Environmental, 1), growing between 20 and 45oC and flourishing at 37oC, and BAP

media will be used for future testing given its encouragement for competition. A few beneficial

adjustments to the procedures would be serial diluting and plating all media at the same time, and

incubating two sets of serial dilution plates of the same media in two different temperatures. This will

make comparing the CFU per gram of soil much clearer as time will stay as an independent variable

while the temperature and media type can be cross referenced as dependent variables.

Conclusion:

This lab and the experiments performed as a part of it has allowed for the conclusion that the Colour

Lake Sediment is a sandy loam type soil with a pH of 5 that grows well in BAP media at 25oC. This

knowledge allowed for the selection of the most adequate media, BAP and G. LB, to be used in the

picking and patching portion of the lab. The main objectives of characterizing the soil sample in both

qualitative and quantitative methods were reached – the CFU/g of the CLS sample is now known on two

different temperatures of three types of media, and a high diversity of colonies from the soil sample was

observed. The hypothesis that the CLS soil sample is diverse and contains billions of cells per gram is

confirmed, save that the order of cells observed were in the millions. The end result of gaining a better

understanding of the unknown soil sample was successfully achieved. The deepened understanding of

the CLS soil sample and its interactions with different media types is greatly beneficial to the next step of

antibiotic activity screening.

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Honesty Statement:

I certify that this lab report represents solely my own efforts. I am aware of University regulations about,

and penalties for, plagiarism. Signed by Matthew He on October 26th, 2017.

References:

Brown, R. B. “Soil Texture.” University of Florida IFAS Extension, SL, no. 29, Apr. 1990, pp. 1–8.,

ufdc.ufl.edu/IR00003107/00001.

Bryant, Donald A., and Niels-Ulrik Frigaard. “Prokaryotic photosynthesis and phototrophy illuminated.”

Trends in Microbiology, vol. 14, no. 11, Nov. 2006, pp. 488–496., doi:10.1016/j.tim.2006.09.001.

Hernandez, Simon, et al. Small world initiative. Small World Initiative Press, 2016.

Miller, J H. “Experiments in molecular genetics.” 1972.

Schloss, Patrick David, and Jo Handelsman. “Toward a census of bacteria in soil.” PLoS Computational

Biology, preprint, no. 2006, 21 July 2006, doi:10.1371/journal.pcbi.0020092.eor.

Schmieder, Robert, and Robert Edwards. “Insights into antibiotic resistance through metagenomic

approaches.” Future Microbiology, vol. 7, no. 1, 2012, pp. 73–89., doi:10.2217/fmb.11.135.

Thien, Steve, and DeAnn Presley. “Estimating Soil Texture by Feel.” Kansas State University Bookstore,

Kansas State University, Sept. 2008, www.bookstore.ksre.ksu.edu/pubs/MF2852.pdf

“Understanding Nosocomial Risk Factors.” ALS Environmental, ALS Scandinavia, 2014,

www.alsenvironmental.co.uk/media-uk/pdf/datasheets/microlp/als_micro_eskape_pathogens_

2014.pdf
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Appendix A