1. Bacterial diversity and composition in the marine environment Sen-Lin Tang ( 湯森林 ), Pei-Wen Chiang ( 江培汶 ), Ching-Hung Tseng ( 曾景鴻 ) Biodiversity Research Center, Academia Sinica 中央研究院生物多樣性研究中心 08.2014

2. Outline Preparation of seawater samples – Introduction to sampling method Isolation of microbes in the seawater samples – Introduction to basic culture techniques – Specific bacterial groups Culture-independent technique for detection of microbe – Introduction to culture-independent technique

3. PREPARATION OF THE SEAWATER SAMPLES Day 1

4. Sample collection 82.5K Seawater samples are isolated from: 1.Coastal water as a control 2.Fresh water 3.Seawater from fish pond 4.Seawater from aquarium Seawater samples are isolated from: 1.Coastal water as a control 2.Fresh water 3.Seawater from fish pond 4.Seawater from aquarium Coastal water Fresh water Fish pond Aquarium (A)(B)(C) Coastal water Fish pond Coastal water

5. Practice Experiment 1: To collect the seawater samples – Students are divided into three groups (groups A, B, C) – Seawater collection bottle Coastal water Fresh water Fish pond Aquarium (A)(B)(C) Coastal water

6. ISOLATION OF MICROBES IN SEAWATER SAMPLES

7. The basic techniques for isolating, cultivating and charactering microbes Equipment and materials Pure culture techniques Media Autoclave Culture tubes Petri dishes Wire loops and needles Spreader Pipettes Incubators (or waterbaths) Refrigerators Broth Semisolid Solid Plate streaking Plate pouring Plate spreading Isolation of pure culture Transfer instruments Cultivation chambers Photo from COPAN Agar slant Agar deep Agar plate

8. Cultivation of microorganisms Nutritional needs – Carbon, nitrogen, metallic elements(e.g., Ca ++, Mg ++ …), nonmetallic element (e.g., sulfur), water, vitamins, energy…etc. Physical factors – Temperature, pH, oxygen.

9. Serial dilution-agar plate procedure To quantitate viable cells. Isolation of discrete colonies that can be subcultured into pure cultures. Serial dilution

10. Practice Experiment 2: Microbial enumeration and isolation – Learn to serial dilution and spreading plate 10 0 〜 10 -3 – The samples collected from Experiment 1 Original sample (10 0 ) 1:10 (10 -1 )1:100 (10 -2 )1:1000 (10 -3 )

11. Use of Differential and selective media Differential media – These can distinguish among morphologically and biochemically related groups of organism. – Chemical compounds that, produce a characteristic changes or growth patterns, which permits differentiation. Selective media – These media are used to select (isolate) specific groups of bacteria. – Chemical compounds that inhibit the growth of one type of bacteria while permitting growth of another.

12. Example: Mannitol salt agar A high salt concentration (7.5% NaCl) which is inhibitory to the most bacteria other than Staphylococci ---Selection The carbohydrate mannitol and the indicator phenol red for detecting acid produced by mannitol fermenting Staphylococci ---Differentiation Coagulase-positive StaphylococciCoagulase-positive Staphylococci produce yellow colonies with yellow zones, whereas coagulase-negative Staphylococci produce small pink or red colonies with no color change to the medium.coagulase-negative Staphylococci

13. Thiosulfate Citrate Bile Salts Sucrose Agar (TCBS) is recommended for use in the selective isolation of vibrios. ( 硫代硫酸鹽 - 檸檬酸鹽 - 膽鹽 - 蔗糖洋菜培養基 ) Isolation of Vibrio strains from coastal water Incubate plates, protected from light, at 35 ± 2°C in an aerobic atmosphere for 18-24 h. Typical colonial morphology on TCBS Agar is as follows: V. cholerae........................................Large yellow colonies V. parahaemolyticus........................Colonies with blue to green centers Inhibition of gram-positive bacteria Sucrose is included as a fermentable carbohydrate for the metabolism of vibrios. Thymol blue and bromthymol blue are indicators of pH changes.

14. Isolation of coliforms from coastal water m Endo Agar LES is used for enumerating coliforms in water. Inhibition of gram-positive bacteria Coliform bacteria ferment the lactose, producing a green metallic sheen. Basic fuchsin is a pH indicator. light pink/colorless=no fermentation, metallic green sheen/greenish=extreme lactose fermentor Incubate plates, produce a red colony with a metallic (golden) sheen within 24 hours incubation at 35°C. Cultural Response: Escherichia coli 25922…………………………..Red with sheen Salmonella enterica………………………………Pink

15. Practice Experiment 3: Selection of specific bacterial groups – To use selective media to discover a specific microbial group (Vibrio spp. and coliforms) – The samples collected from Experiment 1

16. DETERMINATION OF MICROBIAL CONCENTRATION IN SEAWATER Day 2

17. 10 -1 10 -2 10 -3 10 -4 10 -5 Original seawater 10 0 Too numerous to count (more than 300) --- TNTC Too few to count (fewer than 30) --- TFTC

18. How to calculate the colony forming unit? Statistically valid plate counts are only obtained from bacterial cell dilutions that yield between 30 and 300 colonies. Number of cells per ml = number of colonies X dilution factor. Example: – Colonies per plate = 50 – Dilution factor = 1.0 X 10 5 (1*100,000) – Volume of dilution added to plate = 0.1ml – 50 X 100,000 = 5,000,000(5*10 6 ) cells/0.1ml = 50,000,000(5*10 7 ) CFUs/ml

19. Practice Experiment 4: Determination of microbial population sizes – The serial dilution agar plates from Experiment 2 – Record your observations and calculated bacterial counts per ml of sample in the chart

20. THANK FOR YOUR ATTENTION