safty learning excellence 1. total marks:100 marks sessional exams: 50 marks class room tests: 2 @...

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Mic-240:LABORATORY SKILLS

IN MICROBIOLOGY

(Dr. Sarfaraz Hadi)

SAFTY EXCELLENCE

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Mic-240: Laboratory skills in microbiology

Marks allocation

Total marks: 100 marks

Sessional Exams: 50 marks

class room tests: 2 @ 10 = 20

Lab Exercises: 2 @ 15 = 30

Final exam: 50 marks

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Scope of the present course module

- Hazards in microbiology lab- Precautions to be taken in a microbiology lab- Preparation of stock solutions- Preparation of media- Sterilization of media and solutions- Culturing bacteria and fungi

- Use of laminar air flow hood- common media used for fungal and bacterial culture

- Isolation of pathogenic fungi and bacteria from plant material

- Isolation of comensal bacteria from human body- Microscopy- Staining and observation of fungi- Gram staining of bacteria- Extraction/isolation of DNA

- Basic concept of polymerase chain reaction (PCR)

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Equipments used in a microbiology lab

- Air sampler- Autoclave- Laminar air flow hood

- Bead sterilizer/ Bunsen’s flame- Hand instruments

- Incubator- Colony counter- Microscope- Thermal cycler (PCR)- Distillation unit- Precision balance- Heater/microwave oven- Stirrer - Lab ware: glass ware/plastic ware/petriplates

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Hazards in a microbiology lab.

Microbes:- Bacteria

- Fungi

Chemicals:- Acids and alkalis – HCl, NaOH etc.

- Fungicides and antibiotics

- Other toxic substances - HgCl2 ,

Fumes:- Chlorine, formalin etc.

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Hazards in a microbiology lab.Cont…

- UV light

- Hot materials – media, instruments etc.

- High pressure steam (autoclave)

- Glass splinters, scalpel blades etc.

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Precautions(Good laboratory practices)

1. Wear a lab coat to protect clothing from contamination or accidental spillage of harmful chemicals and staining solutions.

2. Wash your hands properly before and after the work.

3. Use face mask and cap during the work.

4. Clean the work area (laboratory bench) with a recommended disinfectant such as 5% Lysol or 5% phenol before and after each work session.

5. Never eat, drink, or smoke in the laboratory.

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PrecautionsCont…

6. Keep the laboratory work area free from articles not in use and return all reagents, cultures, and glassware back to their appropriate places.

7. Keep your hands away from your mouth and eyes while in the laboratory. Do not place anything such as pencils, food, and fingers in your mouth.

8. Avoid contamination of benches, floor, and wastebaskets.

9. Place all discarded cultures, infectious materials, used glass slides and contaminated glassware into receptacles meant for special disposal.

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PrecautionsCont…

10. Place all used or contaminated pipettes in appropriate glass jar filled with a disinfectant such as 5% phenol.

11. If infectious material is spilled accidentally, cover it immediately with a disinfectant such as 5% Lysol or 5% phenol and notify your seniors at once.

12. Do not move with a loop or pipette containing infectious material through the laboratory. Flame wire loops and needles before and after use.

13. Label all your experimental material properly.

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Stock solutions

- Working solution- Stock solution- Dilution of stock solution

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Percent solutions

- Strength of the solution: percentage or parts per hundred.

- Weight in grams – volume 100 ml.

- Same percentage: variation in volume → variation in

weight.

Amount of solute in grams = Required %age × required volume (in liter) × 10

Example: Prepare10% solution of NaCl in 25 ml volume

Amount of NaCl in gram = 10 × 0.025 × 10 = 2.5 g

-Weigh 2.5 g NaCl ,

- Dissolve in ≈ 20 ml water,

- Make volume 25 ml.

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Percent solutions

-Course: Mic-240 (Laboratory skills)

Lab Exercise – 1

Date……………

Name of student: …………………………………………….

Exercise – 1:

Prepare………. % solution of sodium chloride (NaCl) to make final

volume ……………..ml.

Objective:

Materials used:

1.

2.

3.

4.

Procedures:

1. Calculated the amount of Nacl required according to the formula:

Amount of solute in g = Required %age x required volume in liter x 10

2.

3.

4.

Results:

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ppm (parts per million) solutions(mg per liter)

Formula:

Amount of solute in mg = Required concentration× required volume (in

liter)

Example: Prepare100 ppm solution of NaCl in 250 ml volume

Amount of NaCl in mg = 100 × 0.25 = 25 mg

-Weigh 25 mg NaCl ,



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Recording experimentscont…

Exercise :

Prepare……………. ppm solution of sodium chloride (NaCl) to make final volume …………….. ml.

Objective:

To prepare……………. ppm solution of sodium chloride (NaCl) to make final volume …………….. ml.

Materials used:

1. Sodium chloride (NaCl) – lab grade, manufactured by ……………….

2. Precision weighing balance, weighing boat, spatula

3. volumetric flask of known volume, glass funnel

4. distilled water, wash bottle

Procedures:

- Calculated the weight of NaCl required according to the formula:

Weight of NaCl required = concentration required in ppm x volume required in liter.

- Precisely weighed NaCl in a weighing boat on a precision balance using a spatula.

- Transferred entire quantity of NaCl into the volumetric flask by washing the boat with dist. Water.

- Poured about half the final volume distilled water and dissolved NaCl completely

- Made the final volume to the mark of volumetric flask (bottom of the meniscus touching the mark).

Results:

Prepared ………….. ppm solution of NaCl to make final volume ………………. ml.

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ppm (parts per million) solutions(mg per liter)

- Course: Mic-240 (Laboratory skills)

Lab Exercise – 2

Date……………

Name of student: …………………………………………….

Exercise - 2:

Prepare……… ppm solution of sodium chloride to make final volume

……………..ml.

Objective:

Materials used:

1.

2.

3.

4.

Procedures:

1. Calculated the amount of Nacl required according to the formula: Amount of solute in mg = Required concn. (ppm) x required volume in liter 2.

3.

4.

Results:

Course: Mic-240 (Laboratory skills)

Lab Exercise – 2

Date……………

Name of student: …………………………………………….

Exercise - 2:

Prepare……… ppm solution of sodium chloride to make final volume

……………..ml.

Objective:

Materials used:

1.

2.

3.

4.

Procedures:

1. Calculated the amount of Nacl required according to the formula: Amount of solute in mg = Required concn. (ppm) x required volume in liter 2.

3.

4.

Results:

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Molar solutions

Formula weight (FW)/Molecular weight (MW) in grams = 1mole

→ 6.0021415x1023 molecules (Avogadro’s number)

→ Dissolved per liter = 1 Molar (1M) solution = 1 mole per liter

(K=39; Na=23; Cl=35; KCl= 74.55; NaCl= 58.44)

Amount of solute in grams = Required Molarity × FW × required

volume (in liter)

Example: Prepare 200 mM (0.2M) solution of NaCl (FW: 58.44) in 25 ml

Amount of NaCl in grams = 0.2 × 58.44 × 0.025 = 0.292 g (292.2mg)

-Weigh 0.292 g NaCl ,



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Lab Exercise – 3

Date……………

Name of student: …………………………………………….

Exercise - 3:

Prepare……… M solution of sodium chloride (FW = 58.44)to make

final volume ……………..ml.

Objective:

Materials used:

1.

2.

3.

4.

Procedures:

1. Calculated the amount of Nacl required according to the formula: Amount of solute in g = Required Molarity x FW x required volume in liter 2.

3.

4.

Results:

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Usage (dilution) of concentrated solutions

- Stock solutions: high concentration.

- Working solution: lower concentration.

- Transfer portion of stock solution to working solution.

S1 × V1 = S2 × V2 where,

S1: Strength of stock solution (in ppm or %age, or Molarity)

S2: Strength of working solution (in same unit as S1)

V1: Volume of stock solution to be used (in ml or liter)

V2: Volume of working solution (in same unit as V1)

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Usage (dilution) of concentrated solutionscont…

Example: find the volume of 2M stock solution to be used for making 200mM (0.2M) working solution of 50 ml volume.

S1 × V1 = S2 × V2

Strength of stock (2M) x volume required = Strength of

working solution (0.2M) x volume of working solution (50ml)

Volume required = (0.2 x 50) ÷ 2

= 10 ÷ 2 = 5 ml

(Same procedure to be followed for %age, ppm, or ppb solutions)

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Lab exercise – 4Dilution of ppm solution

-


Lab Exercise – 4

Date……………

Name of student: …………………………………………….

Exercise – 4:

Dilute ..………. ppm stock solution of sodium chloride (NaCl) to

…………….. ppm in ……………….. ml final volume.

Objective:

Materials used:

1.

2.

3.

4.

Procedures:

1. Calculated the volume of stock solution required according to the formula:

S1 x V1 = S2 x V2 → V1 = {S2 x V2} ÷ S1

Where: S1 = strength of stock solution; S2 = Strength of final solution; V1 = volume of stock required; V2 = volume of final solution

2.

3.

4.

Results:

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Lab exercise – 5Dilution of molar solution


Lab Exercise – 5

Date……………

Name of student: …………………………………………….

Exercise – 5:

Dilute ..………. M stock solution of sodium chloride (NaCl) to

…………….. M in ……………….. ml final volume.

Objective:

Materials used:

1.

2.

3.

4.

Procedures:


M1 x V1 = M2 x V2 → V1 = {M2 x M2} ÷ S1

Where: M1 = strength of stock solution; M2 = Strength of final solution; V1 = volume of stock required; V2 = volume of final solution

2.

3.

4.

Results:

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Stock solutions with strength of solute in multiples

Concentration → Multiple of known amount per liter

Example:

Required amount in working solution:

Solute x 2 g/l

Solute y 3 g/l

Solute z 4 g/l

5X Strength of stock solution (in 1 liter):

Solute x 2 g/l x 5 = 10 g

Solute y 3 g/l x 5 = 15 g

Solute z 4 g/l x 5 = 20 g

Water ……………………. to make 1 liter

Amount of solute in stock solutions = conc. of solute in working solution × Strength multiple of stock × volume of stock solution (same unit as for working solution).

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Stock solutions with strength of solute in multipleCont…

Example: Medium / working solution - NaCl: 2 g/l

Make 5x solution – 50 ml

Amount of NaCl required (in gram) = 2g/l × 5 × 0.05 liter

= 0.5 g

Usage: 5x soln. – 50 ml

Vol. of stock soln. to use (in ml):

Vol. of working soln. ÷ Strength of stock solution (in x)

Example: volume of above stock required for 100 ml working soln.?100 ml ÷ 5x = 20 ml

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Lab exercise – 6Dilution of multiple strength solution


Lab Exercise – 6

Date……………

Name of student: …………………………………………….

Exercise – 6:

Make ………….ml medium containing 1x strength of sodium chloride

from x stock solution of ………………ml original volume.

Objective:

Materials used:

1.

2.

3.

4.

Procedures:


Vol. of stock soln. to use (in ml):

Strength of stock soln{Vol of working soln ÷ Original vol of stock soln} × 10 (in x) (in ml) (in ml).

3.

4.

5.

Results:

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In-vitro culture of microorganisms(Fungi/Bacteria)

1. In-vitro: Inside the glass (Latin) – (tube – petri plate – flask)

2. Culture: Growth under controlled conditions (sterility – nutrition – temperature – humidity – light etc.)

3. All work to be performed under sterile conditions – on a laminar air flow hood – to avoid contamination

4. Hand instruments (loop – forceps – needle – scalpel etc.) to be sterilized in an autoclave or with a flame/bead sterilizer.

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5. Culture media

1. Nutrition

- Carbon - sucrose, glucose etc.

- Nitrogen (N) – amino acids

- Minerals – (Natural sources/synthetic form)

- Phosphorus (P), Sulfur (S), Iron (Fe), Potassium (K), Copper (Cu), etc.

- Essential amino acids

- Vitamins

2. Support system/matrix – solid (Agar) or liquid

3. Aeration

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5. Culture mediacont…

Types of media:

1. Defined media: components are known/controlled

2. Undefined/complex media: components supplied through natural sources - yeast extract, peptone etc.

Solidification/gelling of the medium: agar, phytagel, agarose

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Preparation of media:1. Weight and dissolve media components in water

one by one (add agar in the last)

2. Adjust pH of the medium (if required)

3. Sterilize the medium – Autoclaving: 20 psi/1.4 kg/cm2

20 min.

4. Allow to cool down to ~60 °C

5. Pour in sterilized petri plates or tubes

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7. When making medium in unsterilized tubes:

- Pour the medium in tubes before autoclaving

- autoclave the tubes

8. Tilt the tubes with molten agar medium by 30-35° to make slants

- To increase surface area

- To make handling more convenient

- To make a gradient of thickness and moisture

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Frequently used media in microbiology lab

PDA (Potato Dextrose Agar) medium:

- Take 300 g potato and wash well with tap water

- Cut the tubers to small pieces (~ 1 cm) without peeling

- Boil in 1l distilled water till it remains 700 – 800 ml

- Sieve the content

- Add and dissolve 20 g dextrose (glucose)

- Add 20 g agar and make volume 1000 ml

- Autoclave at 20 psi for 20 minutes

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Lab Exercise

Exercise :

Prepare 250 ml PDA (Potato Dextrose Agar) medium , pour and solidify it in sterilized petri plates.

Introduction/Objective:

To Prepare 250 ml PDA (Potato Dextrose Agar) medium , pour and solidify it in sterilized petri plates.

Materials used:

1. Potato - ~100 g; knife/scalpel.

2. Dextrose (glucose), Agar powder, distilled water.

3. Weighing balance, conical flask, autoclave.

4. Laminar air flow hood, sterilized (disposable) petri plates

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Lab Exercise-Cont…

Methods/Procedures:

1. Washed potatoes with tap water; cut into small pieces

(~ 1 cm).

2. Weighed 100 g potato pieces, transferred into 500 ml flask, added 250 ml dist. water.

3. Boiled in autoclave at 20 psi for 15 min.

4. Sieved the broth in another 500 ml flask.

5. Added, dissolved 5 g glucose + 5 g agar powder.

6. Autoclaved: 20 psi – 15 min; allowed to cool to ~ 60 °C

7. Poured in petri plates in laminar flow, sealed with parafilm.

Results:

Prepared 250 ml PDA medium as above.

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Lab Exercise – 7

Date……………

Name of student: …………………………………………….

Exercise - 7:

Prepare 250 ml PDA (Potato Dextrose Agar) medium, pour and

solidify it in sterilized petri plates.

Objective:

Materials used:

Methods/Procedures:

Results:

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- Media for culturing fungi- Media for culturing bacteria- - Defined media- - Undefined/complex media- V8 medium- Nutrient agar- Nutrient broth- LB broth- LB agar- Sabouraud Medium- Zapek-Dox

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Media for bacterial culture

Nutrient agar:Peptone………………….5g

Meat extract……………..3g

Agar……………………..15g

Distilled water to make….1 liter

- Weigh the components/Premade medium- Mix in distilled water (~ 800 ml)- Make the final volume 1 liter- Autoclave at 20 psi for 20 minutes- Pour in petri plates on laminar flow

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4. In-vitro culture of microorganismscont…

Laminar air flow hood/bench

1. Laminar air flow hood/bench (laminar: in layers)

Pre filter

Blower

HEPA filter (high efficiency particulate air filter)

Pressure gauge (20 mm water/ 200 pa - Pascal)

light source

UV radiation source (sterilization)

Provision for gas pipe/electrical power

2. Air velocity: 0.4 – 0.5 m/sec – 90 FPM (feet per minute)

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Cleaning the work bench

1. Laminar flow hood or work bench

2. Immediately before and after the work

3. 70% alcohol 4. Lacto phenol 5. Commercial preparation

6. Hands should also be regularly wiped with 70% alcohol or a disinfectant before and during the work.

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3. Sterilization

- Complete destruction or elimination of all forms of microorganisms from a material by physical or chemical methods.

- Media and solutions

- Hand instruments

- glassware, and petri plates etc.

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3. Sterilization Cont…

1. Application of dry heat

Direct flaming – (Hand instruments: Loops, Forceps, Scalpel etc.)

Glass bead sterilizer – (Ditto)

Hot air oven – (Glassware, hand instruments etc.)

2. Application of moist heat Autoclaving (steam under pressure)- 20 pounds per square inch (psi)/ 1.4 kg per sq. cm

- 121 °C

- (Media, glassware, lab coats etc.)

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3. SterilizationCont

Application of moist heat Autoclaving (steam under pressure)

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3. SterilizationCont…

3. Irradiation (200-280 nm)Ultra violet (UV) radiation: 200-280 nm (nano meter)

- (Hospitals, OTs, Labs etc.)

Gamma radiation (γ): 2-10 pm (peco meter)

- (Food material, Lab ware, Liquids etc.)

4. Chemical treatment

Mercuric chloride - (Host material etc.)

Sodium hypochlorite - (Host material etc)

Formalin, phenols - (Work bench, floor etc.)

Ethanol - (Instruments, etc.)

Isopropanol - (Instruments, etc)

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3. SterilizationCont…

5. Filter sterilization Asbestos filters, membrane filters

and sintered glass filters: pore size: 0.1µm , 0.22µm, 0.4µm –

10 µm

- (Media, Heat labile solutions,)

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3. Sterilization Cont…

Filtration

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Cleaning glassware

1. Use sterilized disposable plastic ware Or

2. Submerge the glassware in a cleaning solution: • Potassium dichromate ................... ............ 2 g• Distilled water ................................ ......... 200

ml• Dissolve dichromate in water• when cool, add very slowly:• Concentrated sulfuric acid ........................

..9 parts• Potassium dichromate solution (2%)…... ..1

part

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Isolation of fungi

Materials required:

- Tape water

- Ethyl alcohol

- Scalpel/blade

- PDA plates

Procedures/Methods:

- Clean/wash the source thoroughly

- Wipe the surface with alcohol to kill external organisms

- Cut a thin slice of tissue/material from the infected source

- Plate it on PDA in a petri plate

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Isolation of bacteria

1. From infected plant material: Same as for isolation of fungi

2. From human body: (Mouth, Nose, ears etc.)

Materials required:

- Cotton buds

- Nutrient broth/Nutrient agar

Procedure/Methods:

- Rub the bud on the probable site of bacterial colonization

- Shake the bud in nutrient broth/streak on nutrient agar

- Incubate at appropriate temperature (28-37 °C) for appropriate length of time (8-24 h)

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Isolation of bacteriaCont…2

3. From human body: (blood, sputum, urine, other fluids)

Materials required:

- Pipette

- Nutrient broth/Nutrient agar

Procedure/Methods:

- Pipette a small portion in nutrient broth or plate on nutrient agar

- Incubate at appropriate temperature (28-37 °C) for appropriate length of time (8-24 h)

- Make pure cultures, if needed (contamination/mixture)

- Observe and identify the cultures under a microscope

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Microscopy

Microscopy: Technique of using microscopes for viewing magnified image of small objects.

Microscope: An instrument to observe small objects not visible to naked eye.

→ Greek: ‘micros’ = small + ‘skopein’ = to look

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MicroscopyCont…2

Types of microscopes:

- Optical or Light microscopes:

Use beam of light through single or multiple lenses - samples can be directly viewed by the eye.

- Simple microscope: single lens

- Compound microscope: multiple/many lenses

- Bright field microscope: most common

- Phase contrast microscope: refractive index

- Fluorescent microscope: fluorescent dye

- Digital microscope: image on monitor

- Stereo zoom: external morphology – 3D

52

MicroscopyCont…3

- Electron microscopes:Use beam of electrons through magnifying magnets – Image can be obtained on monitor or film only.

- Transmission Electron Microscope (TEM)

- Scanning Electron Microscope (SEM)

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MicroscopyCont…4

1 = Ocular/Eye pieces

2 = Objective holder

3 = Objectives

4 = Coarse adjustment

5 = Fine adjustment

6 = Stage/Platform

7 = Light source/condenser

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MicroscopyCont…4

Principle of common microscope:

Magnification of microscope =Mag. eye piece x Mag. objective

10x x 40x = 40010x x 100x = 1000x

Object---------------------------------

Image---------------------------------

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Gram staining

- Hans Christian Gram: 1884

(Danish microbiologist)- Staining procedure with crystal violet:

- Purple/blue colored cell: Gram +ve

- Pink colored cells: Gram -ve

- Subsequently:

- Purple & pink cells same population: Gram variable

- Cells without stain: Gram indeterminate

Crystal violet binds to peptoglycan component of cell wall

- Gram +ve bacteria: Thick peptoglycan layer (50-90%)

- Gram –ve bacteria: Thin peptoglycan layer (8-10%)

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Wall structure of Gram +ve & Gram –ve bacteria

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Gram staining procedure

1. Put bacterial cultures on the slide

2. Heat-fix the cells by carefully passing the slide over a flame

3. Add a drop of crystal violet (primary stain) – incubate: 1minute

4. Rinse the slide with gentle stream of water: ~ 5 seconds

5. Add Grams iodine (mordant – binds crystal violet to bacterial cell wall)

6. Rinse the slide with alcohol or acetone: ~ 3 seconds (continued presence of alcohol may remove stain from G+ve cells also)

7. Add safranine (secondary stain) – incubate: 1 minute

8. Rinse the slide with gentle stream of water: ~ 5 seconds

9. Add a drop of glycerol and lay a cover slip

10. Observe under the microscope

- Purple/blue colored cells: Gram +ve

- Pink colored cells: Gram -ve

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Stains/reagents for Gram staining

Crystal violet:

( Methyl violet, Gentian violet)

- Crystal violet 300 mg

- Isopropanol 5 ml

- Ethanol/methanol 5 ml

- Dist. water ………… to make: 100 ml

Gram’s iodine:

- Iodine crystals 330 mg

- Potassium iodide 660 mg


Safranin:

- Safranin crystals 400 mg

- Ethanol/methanol 20 ml


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Extraction/isolation of DNA

Location of DNA

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Extraction/isolation of DNAcontd…..

1. Breaking the cell envelop →

By mechanical methods: grinding, sonicating

By chemical methods: degrading cell envelop (detergent)

→

- Cell envelop constituents: lipids, proteins, polysaccharids

- Cytoplasmic contents: lipids, proteins, pigments

- Nuclear envelop: lipids, proteins

- DNA with histones: histones (proteins)

2. Removal of cell wall and cytoplasmic materials →

By detergent or surfactant (lipids)

By protease (proteins)

By phenol-chloroform (proteins & pigments)

3. Removal of RNA: by RNase

4. Precipitation of DNA:

By sodium acetate, ammonium acetate, ethtyl alcohol

5. Dissolution of DNA in Tris-EDTA or demineralized water

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Extraction/isolation of DNAcontd…..

Isolation of fungal DNA- Grind ~500 mg mycelium in ~250 mg sand- + 600 -l SDS extraction buffer - Transfer to 1.5 ml tube- Heat in water bath: 60°C – 30 min - Add 150 -l 3M sodium acetate - shake by inversion - incubate: RT – 20 min- Spin: 10,000 rpm – 5 min- Collect upper phase- Add 2/3rd volumes (150-l) cold isopropanol – incubate: RT – 15 min- Add 1/2 volumes (300 -l) ethanol – incubate: RT – 5 min- Spin: 10,000 rpm – 5 min – discard supernatant- Wash pellet with 70% ethanol- Spin: 10,000 rpm – 5 min – discard supernatant- Air-dry pellet- Re-suspend DNA in TE

_________________________________________________- - Wash the sand thoroughly 3 times with tap water

- Wash with 70% ethanol- Wash with distilled water once- Autoclave in dist. Water- Decant dist. water and dry in oven

safty learning excellence 1. total marks:100 marks sessional exams: 50 marks class room tests: 2 @...

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