student lab guide - 2 hour labs - part 1

7/31/2019 Student Lab Guide - 2 Hour Labs - Part 1

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1Laboratory Activities (2 hour labs)Biology 2230 Fall, 2011

Lab # Activity Lab bookExercise #

1 IntroductionCulture Media PreparationBrightfield Microscopy

-----181

2 Culture Media Preparation (pouring plates)Survey of MicroorganismsUbiquity of Bacteria, bacterial growth - Inoculation

185, 6, 76

3 Ubiquity of Bacteria, bacterial growth - EvaluationAseptic Technique - InoculationPure Culture Techniques Inoculation

689

4 Aseptic Technique EvaluationPure Culture Techniques EvaluationSmear PreparationSimple StainingNegative StainingGram Staining - Preparation

8910111214

5 Gram StainingSpore Staining

1415

6 Flagella StainsCapsular StainsAcid-Fast StainsOPEN LAB

-----1316-----

7 LAB EXAM # 1 -----

8 Culture Media PreparationDilution ProblemsCulture Media Preparation (pouring plates)

18-----18

9 Selective and Differential Media - InoculationBacterial Counts in Food InoculationBacteriophage Preparation

-----4421

10 Selective and Differential Media EvaluationBacterial Counts in Food EvaluationBacteriophage Inoculations

-----4421

11 Bacteriophage EvaluationsMorphological Study of Unknown Bacterium InoculationCultural Characteristics InoculationOxidation and Fermentation Tests InoculationAntibiotic Sensitivity Testing Preparation

2134353631

12 Morphological Study of Unknown Bacterium EvaluationCultural Characteristics EvaluationOxidation and Fermentation Tests EvaluationBergeys Manual (ID Unknown Bacterium)Antibiotic Sensitivity Testing Inoculation

3435363931

13 Antibiotic Sensitivity Testing EvaluationLab CleanupOPEN LAB

31

14 LAB EXAM # 2 -----


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2

Microscope Storage Procedure(Columbia Lab)

The following steps must be followed each timethe microscope is put away.

1. Turn down the lamp rheostat (dimmer knob).

2. Turn off the lamp.

3. Rotate the nosepiece to the Scanning (4X)objective.

4. Remove the slide from the stage.

5. Move the stage all the way down.

6. If immersion oil is used, use lens paper to wipeoil off the following:a. Oil immersion (100X) objectiveb. High-dry (40X) objectives.c. Staged. Slides

Use a fresh sheet of paper for each objective.

7. Wrap cord snugly around arm below the stage.

8. Replace the dustcover.


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3

Microscope Storage Procedure (Franklin Lab)

The following steps must be followed each time themicroscope is put away in the cabinet.





5. If immersion oil is used, use lens paper to wipeoil off the following:

a. Oil immersion (100X) objectiveb. High-dry (40X) objectives.

c. Staged. Slides

6. Rotate the Scanning (4X) objective into position.

7. Wrap cord snugly around arm above the stage.

8. Return microscope to its correct numbered spotin the cabinet with the back facing outward.The number is on the lower right rear of themicroscope.


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Microscope Storage Procedure(Lawrenceburg Lab)

The following steps must be followed each timethe microscope is put away.





5. If immersion oil is used, use lens paper to wipeoil off the following:

e. Oil immersion (100X) objective

f. High-dry (40X) objectives.g. Stageh. Slides

6. Rotate the Scanning (4X) objective into position.

7. Wrap cord snugly around arm above the stage.

8. Replace the dustcover.


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5

Lab 1Introduction

Exercise 18 Culture Media Preparation Part 1

Preparation: read pages 123 130

Background:Culture Media nutrients used to grow cells or microbes

A. Agar nutrients in gel-like formPlatesSlantsDeeps

B. Broth nutrients in liquid form

Procedure:Weigh out then mix media with water in a flask that is at least twice the capacity of the volume of

the water used.If media is an agar, heat to boiling for 1 minute, stirring constantly, to dissolve.

Turn hot plate on "10". Broths do not require heating.Boiling media will foam up, so be ready with a hot pad to quickly remove media from hot plate.Dispense required volume into tubes using a pipet. If preparing plates, just cap flask with foil.Put caps on tubes.For tubes, write 4 labels with class, type of media, and date, and then put on top of caps. Use a

pencil.For flasks used for plates, write 2 labels with class, type of media and date, and then put one on foil

and one on flask.Put the media in the spot designated by the instructor.

Instructor will autoclave media.Rinse out pipettes with water using pipette pump.

Group Media Media Vol. Water Total No. Vol. / tube(g) (mL) Tubes/Plates (mL / tube)

to Make1 Nutrient Broth 0.8 100 20 52 Nutrient Agar Plates 11.5 500 20 253 Nutrient Agar Plates 11.5 500 20 254 Nutrient Agar Slants 1.7 72 18 45 Nutrient Agar Slants 1.7 72 18 46 Nutrient Agar Slants 1.7 72 18 4

Lab Report: none

Know for Exam:Know how to weigh, pipette, mix, and dispense media.Do not have to memorize media recipes.Know how to pour plates.Know the different types of culture media preparations.


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6Exercise 1 Brightfield Microscopy

Preparation: read pages 3 8.

Background:Follow labbook, particularly note the following topics:

A. Carrying microscope

B. Microscope components (Fig. 1.2)Know all componentsKnow objectives scanning, low power, high-dry, and oil immersion

C. Microscope properties1. Magnification

Enlarging imageTotal Mag. = Ocular Mag. x Objective Mag.

2. Resolution

Ability to see fine detail= 0.2 mm for human eye= 0.2 m for compound light microscope

D. Microscope procedures:1. Cleaning

Use lens paper2. Use

Follow directions in bookImmersion oil (Fig. 1.4)

3. Storage

Use checklist at beginning ofhandout

Procedure:1. Observe Blood Smear slide (each pair of students)

a. Go to oil immersion and draw two types of cellsi. Erythrocytes (no nucleus)ii. Leukocytes (nucleus)

2. Instructor checks all microscopes before they are put away.

Lab report: do all except A4

Know for Exam: Microscope components (Fig. 1.2), properties, procedures, storage.How and why immersion oil is used.Information in lab report.How to distinguish erythrocytes and leukocytes on a blood smear slide.

Micrograph by Karen Kendall-Fite

Blood Smear


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Lab 2 Ex 18 Culture Media Preparation

Procedure:Each group label 7 plates NA (nutrient agar) on bottom with a wax pencil & pour.Rinse out empty flasks, remove labels, and put away.

Exercise 6 Ubiquity of Bacteria inoculation


Background:Bacteria are ubiquitous (everywhere) and distributed throughout the biosphere

Procedure: (each group)1. Inoculate one Nutrient Broth

a. Moisten a sterile swab with tap water (is sterile)b. Rub swab on surface of your choicec. Insert swab in brothd. Dispose of swab in biohazard bag

2. Inoculate one platea. Moisten a sterile swab with tap waterb. Rub swab on surface as indicated in table below:

Group Surface or Exposure Method1 Air for 30 minutes (just open plate)2 Hair combed over plate3 Fingers4 Surface of your choice5 Surface of your choice6 Surface of your choice

c. Gently rub swab on the surface of the plated. Dispose of swab in biohazard bage. Label all media with the following information on a paper label

throughout the lab: Section number, Group number, Sample, and Date.f. For tubes, put the label on the glass tube, not the cap.g. For plates, put the label on the bottom of the plate.h. Put media in incubator in spot designated by instructor.i. Always incubate and store plates upside down so condensation does not

fall on the plate and smear bacteria.

Lab Report: none


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8Survey of MicroorganismsExercise 5 Protozoans, Algae, Cy., Exercise 6 Ubiquity of Bacteria, Exercise 7 The Fungi

Preparation: read pages 33, 47, 53

Background:Classification of Microorganisms

Taxonomy science of classificationTaxonomic categories

Domain DistinguishedKingdom KingsPhylum PlayClass ChessOrder OnFamily FineGenus GreenSpecies Silk

Species Similar organisms that can interbreed

Use binomial nomenclature (genus name and specific epithet)Ex: Homo sapiens , Escherichia coli

MicroorganismsDraw the following slides to scale and indicate magnificationEach group use two microscopes and share to speed things up.

I. Domain Eukarya all eukaryotic organisms

A. Kingdom Protista single-celled, or colonial that lack tissuespecialization, eukaryotic (Exercise 5)

1. Subkingdom Protozoa animal like, no cell wallsExample: Genus Paramecium

OBSERVE Paramecium slide (high power)

2. Subkingdom Algae cell walls of cellulose, photosynthetic

Example: Genus Spirogyra OBSERVE Spirogyra slide (low power)

Example: Diatoms

Special category of algaeCell wall consists of two halves that fit togetherlike a box.OBSERVE Mixed Diatoms slide (high-dry)

OBSERVE live sample of Protozoa and AlgaeMake wet mount of pond water or hay infusion

(scanning, low, high-dry)


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9B. Kingdom Fungi eukaryotic, non-photosynthetic, cell walls ofchitin (Exercise 7)

1. MoldsMulticellularFilamentous have hyphaeExample: Genus Rhizopus

OBSERVE Rhizopus sporangia, w.m. slide (lowpower)

2. YeastsSingle-celledGenus Saccharomyces OBSERVE Saccharomyces budding cells, w.m.

(oil immersion)

II. Domain Bacteria prokaryotic

Example: Three different bacteria on one slideOBSERVE Bacteria, three forms; separate smears, Gramstain slides (oil immersion)Find and draw 3 shapes and any arrangements (Fig. 6.1)

III. Domain Archaea prokaryotic, similar size and shapes as bacteriaExample: mixed archaeabacteria

OBSERVE Archaeabacteria Mixed slide (oil immersion)

Procedure:1. Using microscope, observe each of the above indicated slides.

2. Always start at low power and work up to the magnification indicated.3. Draw each organism you see, noting the magnification.4. Instructor checks all microscopes before they are put away.

Lab Report: none

Know for Exam: classifications and characteristics of each microbe listed above.

Micrograph by Karen Kendall-Fite Micrograph by Karen Kendall-Fite

Paramecium Spirogyra


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Saccharomyces


Archaebacteria

Rhizopus Diatoms

Bacteria bacilli Bacteria cocci Bacteria spirals

Micrograph by Jeni Sharpe

Micrograph by Jeni Sharpe Micrograph by Jeni Sharpe

Micrograph by Karen Kendall-Fite Micrograph by Karen Kendall-Fite



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Lab 3Exercise 6 Ubiquity of Bacteria evaluation


Background:Bacteria are solid and mucoid. Molds are fuzzy.

Procedure:Examine plates looking for molds and bacteria.

Lab Report none

Know for Exam: Know proper labeling of inoculated media. Know how to store and incubate plates and why they are stored that way. Be able to distinguish between bacteria and molds on a plate. Know in a general way the results obtained by the class.

Ubiquity of Bacteria ResultsGroup Plate Exposure Method Colony Counts* Broth Exposure Method** Result

Bacteria Mold1

2

3

4

5

6

*Evaluation of plates0 = no growth+ = 1 10 colonies++ = 11 50 colonies+++ = 51 100 colonies++++ = over 100 colonies

**Evaluation of broths0 = clear (no bacterial growth)+ = cloudy (bacterial growth occurred)


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12Exercise 8 Aseptic Technique Inoculation


Background:Aseptic technique handling without contamination

Insures culture is not contaminated by other microbesInsures handler is not contaminated by culture

Procedure:1. Perform broth to broth transfer

a. Use Figures 8.1 and 8.2 except use Bacticinerator instead of burner anddo not flame tubes

b. Use broth from Ubiquity experiment and transfer to a sterile NutrientBroth

c. One transfer per group2. Perform plate to slant transfers as shown by instructor

a. Label 8 Nutrient Agar slants with:

Section numberGroup number (1, 2, 3, 4, 5, 6, Backup, Unknown)BacteriaDate

b. Each group inoculates these 8 slants from one stock culture plate.c. Each person does two transfers by yourself (no buddy system)d. Loop must be flat or will tear agar. Do not rest loop in Bacticinerator.e. Put slants in appropriate place in rack

Lab Report: do next lab

Exercise 9 Pure Culture Techniques Inoculation


Background:Pure culture technique

Method to obtain pure culture from mixed cultureMost commonly used pure culture technique streak plate

Procedure:

Each person does one streak plate using the broth culture from the Ubiquityexercise following instructions on Fig. 9.3 & Fig. 9.4 (Quadrant Streak, Method B)except:

Use Bacticinerator instead of burner.Do not flame the mouth of tubePick up plate from bottom.Hold plate at angle to see where streaked.Include initials on label in addition to the regular information.

Lab Report Do next lab


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Lab 4Exercise 8 Aseptic Techniques Evaluation


Procedure:1. Check all slants and broths2. Put any unsuccessful transfers in Remake rack.3. Put successful slants back in original rack in appropriate place.4. Keep broth for next exercise.

Lab Report: A (1 3), B (1 5)

Know for Exam: Know what aseptic technique is and how to do aseptic transfers. Know lab report material.

Exercise 9 Pure Culture Techniques Evaluation

Preparation: read page 81

Procedure:1. Examine streak plates2. Look for colonies of different color,

size, shape different bacteria

Lab Report: do A1, omit remainder.

Know for Exam: Know what a pure culture technique

is and what it is used for. Know how to do a streak plate.

Exercise 10 Smear Preparation


Background:Smear microbial sample applied to a slide

1. Apply sample with loop2. Air dry3. Fix sample (kills and attaches cells)

Procedure:Follow labbook (Fig. 10.1) except dont wash slidesEach group prepare:

One smear from Aseptic Technique brothOne smear from colony on streak plate

Photograph by Spence Dowlen


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14Lab Report: will do in Lab 6

Know for Exam: Know what a smear is. Understand the steps in making a smear from liquid and solid media.

Exercise 11 Simple Stain (Positive Staining)


Background:Dyes

Dyes are salts + and ionsChromophore is ion that is coloredAuxochrome is ion without color

Positive stainingChromophore is positively charged attracted to negative bacteriaMust fixDRAW

Cells are colored on a light background

Positive StainingSimple Stain use single dye to color a microbeDifferential Stain use two or more dyes to color a microbe

Procedure:Follow procedure shown in Figure 11.1 using two smears previously made.Use Methylene Blue.

Lab Report: will do in Lab 6

Know for Exam: Know how positive staining works. Compare simple versus differential

stains. Know how to do a simple (positive)

stain and the dye we used in the lab. Know lab report material.



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15Exercise 12 Negative Staining


Background:Negative stainingChromophore is negative ion repelled by negative bacteriaDRAW

Cells are transparent on dark backgroundNo fixing

no shrinkage of cell capsules not damaged thin cells more easily seen

Procedure:1. Follow procedure in Figure 12.1 using a large colony and drop of Nigrosin2. Each group does one stain

a. Groups 1-3 use Escherichia coli b. Groups 4-6 use Staphylococcus epidermidis

3. Observe on oil immersion lens.

Lab Report: do in next exercise

Know for exam: Know how negative staining

works and why you might use it. Know how to do a negative stain

and the dye we used in lab. Know Lab report material.

Exercise 14 Gram Staining (preparation)

Procedure:1. Inoculate one nutrient broth with Staphylococcus epidermidis 2. Incubate at 37

C.



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Lab 5Exercise 14 Gram Staining


Background:Differential Staining

1. Primary stain2. Mordant locks in and intensifies primary stain3. Decolorizer4. Counterstain

Gram stainOne type of differential stainDistinguishes gram positive bacteria from gram negative

Gram positive purple

Gram negative pink

Procedure:1. Each group prepare

a. One smear from teeth scrapings (use wooden end of sterile swab)b. One smear from two cultures:

Staphylococcus epidermidis from broth inoculated last lab (use 3 loops)Escherichia coli from stock cultures

2. Using Figure 14.3, Gram stain the two smears.

Lab Report: A 1 (p. 111)

Know for Exam: Know four steps of a differential stain. Know how to do a Gram stain, including dyes used. Know how to interpret a Gram stain. Know lab report material.

Micrographs by Jeni Sharpe


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17Exercise 15 Spore Staining

Preparation: pages 105 106

Background:Endospores

Protective structure formed when conditions unfavorableMost stains will not penetrateSeen in Genus Bacillus and Genus Clostridium

Schaeffer-Fulton methodEndospores greenVegetative cells pink

Differential Stain SummaryGram Stain Endospore Stain

Primary Stain Crystal Violet Malachite GreenMordant Grams Iodine HeatDecolorizer Ethanol WaterCounterstain Safranin Safranin

Procedure:1. Each group prepare one smear of Bacillus subtilis 2. Use the Schaeffer-Fulton method (Fig. 15.1) to perform a spore stain.

Lab Report: (p. 111) A 2

Know for Exam: Know how to do a Schaeffer-Fulton endospore stain, including the dyes used. Know how to interpret this stain. Know what genera of bacteria

produce endospores. Know the lab report material.



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Lab 6Flagella Stains

Preparation: read page 115 116.

Procedure:1. Observe polar and peritrichous flagella arrangements using oil immersion on:

a. Bacteria Flagella Polar Amphitrichous or Spirillum volutans slideb. Proteus vulgaris flagella stain slide

The flagella are fragile and most have broken off. You will need tosearch to find a good example.

Know for Exam: Be able to distinguish flagella arrangements under the microscope.

Micrographs by Jeni Sharpe


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19Exercise 13 Capsular Staining


Background:Capsule

Gelatinous covering outside cell wallClear area around bacteriaCan help protect the bacteria from phagocytosis by immune system cells

Capsule stainDRAW

Procedure:

Observe and draw specimens from Bacterial Capsules slide on oil immersion.

Lab Report: (p. 98-99) B, C, D (omit B5, B6)

Know for Exam: Know what capsules are and be able to identify them on a slide. Know lab report material. Do not have to know how to do stain.



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20Exercise 16 Acid-Fast Staining

Preparation: read pages 109

Background:Mycobacterium has waxy material (mycolic acid) in cell wall that is acid-fast

acid/alcohol will not wash away pink dyeBlue non-acid fastPink acid-fast Genus Mycobacterium

Mycobacterium tuberculosis TBMycobacterium leprae leprosy

Procedure:1. Observe and draw specimens Mycobacterium tuberculosis sputum smear slides

using oil immersion.2. Note that 99% of slide is non-acid-fast (blue). Look for acid fast (pink or red)

Lab Report: (p. 112-114) A3, B, D, E (omit acid fast stain on D)

Know for Exam: Be able to interpret an acid-fast stain when seen on a slide. Do not have to know how to do an acid-fast stain. Know the main genus of bacteria that is acid-fast and why it is acid-fast. Know the lab report material.



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21Summary of Staining

Open Lab

Procedure:1. Observe all slides from previous labs2. Study all information, procedures, and lab reports given in the Lab.

Staining

Positive Staining Negative Staining Special Staining

CapsuleStaining

FlagellaStainin

Differential StainingSimple Staining

GramStaining

MethyleneBlueStaining

Acid-FastStaining

EndosporeStaining

CrystalVioletStaining

SafraninStaining


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Lab 7

Lab Exam # 1

student lab guide - 2 hour labs - part 1

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