bacterial diversity and composition in the marine environment
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
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
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