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

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

PREPARATION OF THE SEAWATER SAMPLES

Day 1

Sample collection

82.5K

Seawater samples are isolated from: 1. Coastal water as a control2. Fresh water3. Seawater from fish pond4. Seawater from aquarium

Coastal water

Fresh water Fish pond Aquarium

(A) (B) (C)

Coastal water Coastal water

Fish pond

Coastal water

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 Coastal water

ISOLATION OF MICROBES IN SEAWATER SAMPLES

The basic techniques for isolating, cultivating and charactering microbes

Equipment and

materials

Pure culture techniques

Media

AutoclaveCulture tubesPetri dishesWire loops and needlesSpreaderPipettesIncubators (or waterbaths)Refrigerators

BrothSemisolidSolid

Plate streaking Plate pouringPlate spreading

Isolation of pure culture

Transfer instruments

Cultivation chambers

Photo from COPAN

Agar slantAgar deepAgar plate

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.

Serial dilution-agar plate procedure

• To quantitate viable cells.

• Isolation of discrete colonies that can be subcultured into pure cultures.

Serial dilution

Practice

• Experiment 2: Microbial enumeration and isolation– Learn to serial dilution and spreading plate• 100〜 10-3

– The samples collected from Experiment 1

Original sample (100) 1:10 (10-1) 1:100 (10-2) 1:1000 (10-3)

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.

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 Staphylococci produce yellow colonies with yellow zones, whereas coagulase-negative Staphylococci produce small pink or red colonies with no color change to the medium.

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 coloniesV. 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 indicatorsof pH changes.

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 sheenSalmonella enterica………………………………Pink

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

DETERMINATION OF MICROBIAL CONCENTRATION IN SEAWATER

Day 2

10-1 10-2 10-3

10-4 10-5

Original seawater

100

Too numerous to count (more than 300) --- TNTC

Too few to count (fewer than 30) --- TFTC

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 105 (1*100,000)– Volume of dilution added to plate = 0.1ml– 50 X 100,000 = 5,000,000(5*106) cells/0.1ml = 50,000,000(5*107)

CFUs/ml

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

THANK FOR YOUR ATTENTION