Highlights of Bio 200 Summer 2011-2012

(Assessment of Actinomycete Diversity in the Soil Obtained from Mt.

Makiling Forest)

Purification stage

April 4, 2011

• Started first step of purification from tubes

• Streaking for isolation using Actinomycetes Isolation Medium

• All cultures on tubes (from 1000m-100m respectively) should be purified

except for those confirmed to be molds

• Finished 1000m, 900m, and 800m [44 plates]

April 5, 2011

• Inventory

• Media preparation

• Decontamination

April 8, 2011

• Streaked for isolation

• Finished 700m, 600m, and 500m [35 plates]

April 12, 2011

• Streaked for isolation

• Finished 400m and 300m [18 plates]

April 13, 2011

• Broken laminar flowhood

April 14, 2011

• Finished last set of tubes (200m and 100m elevation) [22 plates]

• First step of purification is finished

 • The streaked plates will be incubated in room temperature for 7-14 days

*total number of plates streaked= 119

Observation stage

April 18, 2011

• Observation on the first stage of purification

• Observed isolates from 1000m, 900m, and 800m elevation [18 ok]

April 19, 2011

• Observed isolates from 700m to 100m [47 ok]

• Finished observation [119 plates total; see Table 1]

• All plates ready for next stage of purification was stored in the refrigerator
to avoid overgrowth and contamination

*Total number of plates with isolates= 65

April 20-22, 2011

• Holy week

April 26, 2011

• Media preparation

• Restreaked plates without growth [8R1V1, 8R3G, 8R3L, 8R1H, 1R1G,

8R3H, 8R1G, 8R1E, 1R1E, 1R1D, 8R3J, 8R3E, 9R3B]

May 3, 2011

• Observed restreaked plates

• 11 plates discarded [probably not actinomycete]

• 9 plates ok -> for next stage of purification

*Total number of plates after restreaking= 74

2nd stage of purification

May 4, 2011

• Streaked for isolation

• Finished 33 plates

• 100m, 200m, 300m, 400m, 800m

• 8R3B, 3R3C, 8R3I --- discarded [probably not actinomycete]

May 5, 2011

• Streaked for isolation

• Finished 28 plates

• 1000m, 900m, 700m, 600m, 500m

• Done for 2nd stage of purification [61 plates total]

May 10, 2011

• Observed the plates

• Discarded 1R1C, 1R1I, and 5R1A [probably not actinomycete]

*Total number of isolates purified=58

*Isolates now ready for antibiotic assay

Summary

The first stage of purification started April 4, 2011. Tube cultures were
transferred to plates using streaking for isolation. The total number of plates
(isolates) after the first stage of purification was 119. After the 5-10 days incubation
period, each isolate were observed to determine whether they are pure or not. If the
culture is not pure, it will undergo another stage of purification. Some plates were
contaminated and some had no growth. These plates were re-streaked for it to be
considered for the second stage of purification. The total number of plates with
isolates that can be considered as actinomycete based on their cultural
characteristics were 65. The rest of the isolates were either confirmed to be molds
or other non-actinomycete species.

The second stage of purification started May 4, 2011. The cultures from the
first stage were transferred to another plates to assure purity. Some isolates were
purified for more than two times, but still exhibit mold contamination. The total
number of isolates after the second stage of purification was 58. These isolates
were grown on test tubes to serve as reference for further studies.

Table 1. Cultural characteristics of each isolate after the first stage of purification

Isolat Coloni
e Cultural characteristics/ description es
1R1H no growth, contaminated -
9R3E yeast like, green 1
R2H no growth -
8R3A white, elevated, with opaque center 3
1R2F white-pink, elevated 3
9R2B no growth -
8R1B yellow-green, elevated 3
9R1F cream, flat 2
8R3B mold-like, prob.mold, contaminated 2
8R1V1 no growth -
8R3I cream, elevated contaminated 1
8R2G no growth -
8R3G no growth, contaminated -
1R2A no growth -
8R3L no growth -
1R2I no growth -
1R1E no growth -
1R1A no growth -
8R1H no growth -
9R3A white, crusty 3
9R1B grey, elevated 3
1R1F no growth -
9R1C brown colonies 2
1R2A no growth -
9R1E no growth -
1R1G no growth, contaminated -
1R2G cream, flat 2
9R3A no growth, contaminated -
8R3H no growth -
8R2E no growth 1
8R1G no growth -
1R1I White colonies, contaminated 1
8R1E no growth -
1R1D no growth, contaminated -
9R3C cream/yellow-green, elevated 1
1R1V2 yellow-green, elevated,c 1
8R3J no growth, contaminated -
1R1C white colonies, contaminated 1
8R3K no growth, contaminated -
8R3E no growth -
1R2D green colonies 1
9R3B no growth, contaminated -
1R1V1 yellow-green colonies 1
7R1D no growth, contaminated -
5R1D Contaminated 1
5R3D no growth, contaminated -
7R2C no growth, contaminated -
6R3A pink, dry, elevated colonies 2
6R2F no growth -
6R3B cream-pink, dry colonies 3
6R2D cream, dry, elevated with ring center 1
6R2C white, with transparent surrounding, branching 3
6R2B no growth -
7R3E no growth -
6R1B bright yellow-green, dry, elevated colonies, contaminated 3
6R1F cream-pink, dry, flat colonies 2
7R2A no growth, contaminated -
6R3D no growth -
6R2E white, dry, flat colonies, contaminated 3
brown to light brown, dry, flat, some with dark brown
6R1A center 2
7R3A cream to yellow-green, slimy 2
7R1C white, dry colonies 3
5R1A cream, sticky 2
5R2A no growth, contaminated -
5R3B pink, dry, elevated colonies 3
5R2D no growth -
5R2E pink, dry, elevated colonies, contaminated 2
5R3E pink, dry, elevated colonies 3
5R3A no growth, contaminated -
932C white, transparent, flat colonies 2
7R2D white, transparent, flat colonies 3
5R3C no growth, contaminated -
9R2D no growth -
9R1A no growth, contaminated -
5R2B no growth, contaminated -
7R2B no growth -
7R3B no growth -
5R2F white/cream colonies 2
3R1F no growth -
3R2A cream, dry, flat colonies 2
4R2D bright yellow-green, dry, elevated colonies 3
4R3D no growth, contaminated -
3R1D yellow-green, dry, elevated 3
3R1C no growth -
3R3E light pink, dry, elevated colonies 3
4R3F light brown, transparent, slimy appearance 3
3R3D no growth -
3R3C white, dry, elevated 1
4R2C light pink colonies, contaminated 3
4R2B white, elevated colonies 2
4R2B transparent, star-like colonies, contaminated 1
4R3G yellow-green, dry, elevated colonies, contaminated 3
3R3B white colonies, contaminated 2
4R3B cream, *bact, contaminated 2
3R1A no growth -
4R2A brown, elevated colonies 3
1R1D white, crusy, sub-medial attachment 3
1R2C light pink -
2R2B cream-pink, dry, elevated colonies, contaminated 3
2R1B no growth, contaminated -
1R2E no growth, contaminated -
1R2A no growth, contaminated -
1R2D white, dry, compact colonies, contaminated 1
2R1F cream-pink, dry, elevated colonies, contaminated 3
1R3C no growth, contaminated -
1R3F no growth, contaminated -
2R3D Slimy 3
 2R1D Slimy 2
1R3E white, elevated colonies 3
2R1C white, dry, elevate colonies 3
2R3E no growth, contaminated -
1R3D white/cream, flat colonies 3
2R3A Slimy 3
2R3F yellow-green, dry, elevated colonies 3
2R1A white, elevated colonies, contaminated 2
2R1G cream-pink, dry, elevated colonies, contaminated 3
2R3C Slimy 2
2R1E pink, dry, elevated colonies 3
Legend:
1- 1 to 10 isolates
2- 11-20 isolates
3- 20+ isolates

Assessment of Actinomycete Diversity in the Soil Obtained from Mt. Makiling Forest

Juan Carlo M. Peña

Thesis update submitted as partial fulfilment of the requirements for the degree of
Bachelor of Science in Biology (Microbiology)
May 2011