micro lab mans lsu

of 51BIO 301 MICROBIOLOGYSouthern Leyte State University-Hinunangan Campus

MICROBIOLOGY

LABORATORY

MANUALStudent Manual

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BIO 301 MICROBIOLOGY

Southern Leyte State University-Hinunangan Campus

M A N U A L C O N T E N TS IN T R O DUC T I O N T O MICR O B I O L O G Y L A B O R A T O RY

Welcome to the laboratory component of Microbiology! These laboratory sections have been designed to enhance and “bring to life” the materials that will be covered in lecture by allowing direct observation, experimentation, and application of techniques commonly used when studying the various microorganisms. The student should expect this course to be challenging, informative, and hopefully enjoyable. The latter of the expectations is achieved through preparation (read each exercise prior to your scheduled lab) and active participation in the laboratory exercises.

For the sake of time (and to retain sanity) it is imperative that you prepare before class. Read the introduction to the scheduled exercise, and familiarize yourself with the steps in each activity process (materials and methods section). It is not expected that you understand everything, just be familiar with the activities.

You will be assigned a modified lab write-up as homework for each lab (the format is included in the appendix). The introductory paragraph should include a purpose statement as well as an introduction to the material in the background information. As you will soon realize, experimentation using microbes often requires incubation time for growth in order to make a proper determination from your results. For most exercises results will not be available until the following scheduled class. At that time a record should be made of your results in the given area. Your results sections along with a conclusion paragraph will be assigned as a post-lab.

E X E RC I S E S Exercise 1 – Introduction: Microscopy and Cell types Exercise 2 – Basic Procedure in the Microbiology Lab Exercise 3 – Aseptic Technique and Media PreparationExercise 4 – Morphological examination: Differential Staining Techniques Introduction to Biochemical/Metabolic DifferentiationExercise 5 – Culturing: Media Selection (Defined, Complex, Selective, & Differential) andInoculation techniquesExercise 6 – Media Selection and Metabolic Characterization Continued

Laboratory Practicum I - Basic Laboratory TechniquesLaboratory Practicum II - Identification of an Unknown Bacterium

Exercise 7 – Quantification of Microorganisms – BacteriaExercise 8 – Control of Microorganisms – Testing and Evaluation TechniquesExercise 9 – Transformation of E. coliExercise 10– Parasitology

A PP E ND I X Possible Organisms Chart (for Identification of Unknown)

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EXERCISE 1INTRODUCTION: MICROSCOPY AND CELL TYPES

Introduction:

Microbiology is the study of very small organisms, microorganisms, which can only be viewed with the aid of a microscope. There are several groups of organisms that fit into this category including bacteria, cyanobacteria, fungi, and protists. Within this group there are several species interesting to humans because of their ability to cause disease or their use in the food industry. Many of these microorganisms are unicellular although some are multicellular. These organisms are extremely diverse in cell type, size, color, and reproductive strategy.

When working with microorganisms one easy way to classify them is by their cell type. All cells (including plant and animal cells) can be categorized as either prokaryotic or eukaryotic. The primary difference between these two cell types is the presence of a membrane-bound nucleus. All eukaryotic cells have a membrane-bound nucleus that houses the genetic material (DNA) in addition to other membrane-bound organelles such as mitochondria and chloroplasts. Prokaryotic cells lack a membrane-bound nucleus, their genetic material is located in a particular region of the cell called the nucleoid. In addition to this difference, prokaryotic cells are much smaller than eukaryotic cells. Examples of prokaryotic cells include bacteria and cyanobacteria (photosynthetic prokaryotes). Eukaryotic microorganisms include fungi, protozoa, and algae.

In this lab, we will be getting some first-hand experience with the microorganisms described above. Since the eukaryotic cells are larger than prokaryotic cells, this will be the best place to start. Once you have a feel for these larger cells you are then ready to begin investigating the smaller prokaryotic cells.

• Fungi include unicellular and multicellular eukaryotic organisms. One thing all fungi share is that they are non-motile heterotrophs that absorb dissolved organic material through their cell walls and all but the yeasts metabolize aerobically. We will only be observing yeast in lab. Yeasts are round unicellular microbes that are widely distributed.

• Protists are for the most part unicellular, eukaryotic cells consisting of several groups. The two groups of protists under investigation here are algae and protozoa. The primary difference between these two groups is that algae are photosynthetic while protozoa are described as animal-like because they are heterotrophs (consume other protists, bacteria and detritus). There are several protozoa that cause disease including Plasmodium vivax (malaria) and Giardia lamblia (gastroenteritis).

• Prokaryotes will be the primary focus of the semester. In today’s lab we will observe some of the diversity of this group by looking at both bacterial cells and cyanobacteria. Remember that these cells are much smaller than eukaryotic cells, and lack membrane- bound nuclei.

In order to investigate microorganisms we need to become intimate with our primary tool--the microscope. This invaluable tool allows the viewing of objects/structures that otherwise would go unnoticed by the unaided human eye.

The type of microscope shown below is called the light microscope. Light is conducted through curved lenses in such a way that an object may be viewed larger than its actual size. You might want to label the basic structures and take notes as your instructor goes over the microscope’s structure and function.

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The light microscopes used in this lab are binocular and have ocular lenses with a magnification of 10X. In addition to this magnification there are also four different

objective lenses to choose from – 4X, 10X, 40X, and 100X. Magnification of the object being viewed is the product of the ocular objective multiplied by the lens objective currently in use. For instance when viewing an object on the 4X objective lens, the object is magnified a total of 40X.

Even the highest quality light microscope is limited in its magnification abilities. The highest objective for our microscopes is 100X, which has a magnification of 1000X. With this magnification even the slightest distortion of light would greatly

reduce the quality of the image. In order to conduct the light properly at this objective,

it is it is necessary to place a drop of oil on top of the slide and immerse the lens. The oil

immersion lens (100X) is specially sealed and is the only lens that should be placed in oil. We will learn more about the oil immersion lens

in E2.The importance of proper handling and use of the microscope is vital. You will find this to be especially true as beginning microscopists. It is critical that you clean the microscopes before and after use. Please take notes while your instructor goes over this information with you.

M a t er i a ls N ee d e d :

Blank Slides/Cover slips Iodine10% bleach soln. Cultures:Yeast YogurtGleocapsa

Oscillatoria Anabaena Volvox Spirogyra Amoeba Paramecium

A c ti v iti e s In order to observe the differences between these cell types and become familiar with the microscope you will be preparing several slides primarily using the wet mount technique as described below. In some cases prepared slides may be provided.

Remember to CLEAN your microscope before starting to view your slides. Use a new KimWipe to gently wipe the ocular lenses and then wipe the 10x, 40x, and 100x objective lenses. If there is any excess oil on the microscope be sure and remove that. If you have trouble removing oil, use the microscope cleaner provided.

For each activity below, use the wet mount method to make your slides. Add the drop of iodine as described for those cultures with an (*).1. Add a drop of live culture2. Add a cover slip and then add a drop of iodine* beside the coverslip.3. Observe under the microscope up to 40x and sketch your results in table provided4. Place slide in 10% bleach solution provided


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A c ti v i t y I: F ungus Yeast Observation*

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A c ti v i t y I I: P r o t i s ts A) Protozoa

Amoeba * Paramecium *

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B) Green AlgaeVolvox Spirogyra

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A c ti v i t y I II: P r o k a r y o t e s A) Cyanobacteria

Gleocapsa Anabaena Oscillatoria

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B) Bacteria


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BIO 301 MICROBIOLOGYC) Lactobacillus (yogurt) * No photo available

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Remember to CLEAN your microscope before putting it in the cabinet.

E1 RESULTS:

Eukaryotic CellsType

Sketch on (40x)

Fungi: Yeast

Protist: Amoeba

Protist:Paramecium

Protist: Volvox

Protist: Spirogyra

Prokaryotic Cells

Type Sketch (40x)

Cyanobacteria: Gleocapsa

Cyanobacteria: Oscillatoria

Cyanobacteria: Anabaena

Bacteria: Lactobacillus (yogurt)

E1 Write-upIntroduction paragraph: include a description of the following items from the background information:

• Prokaryotic vs. eukaryotic cells• Introduction of types cells viewed

Submit results from lab manual Conclusion:

• Draw conclusions about the importance of getting familiar with proper microscope technique (for instance proper handling, focusing, and cleaning of the microscope).

• Include a comparison of the prokaryotic and eukaryotic cells observed in this class.

This write-up must be typed and be in your own words.

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EXERCISE 2BASIC PROCEDURE IN THE MICROBIOLOGY LABORATORYIntroduction

Microbes can serve as either our greatest allies or our worst enemies depending on their type and location. This is why studying these organisms is so vital. Methods for studying microbes are as diverse as the groups themselves.

Due to their size and ubiquity, microbes can be a challenge in the laboratory. This unit will focus on the general techniques that are needed in order to achieve good experimental outcomes while protecting the health and safety of everyone. We will be focusing on two important tools in the microbiologist’s tool box—working with bacteria cultures and microscopy.

The first part of the laboratory will introduce you to the materials and methods we routinely use when working with bacterial cultures. The most important technique to learn today is aseptic technique which will be explored in greater detail in E3. In addition to learning this basic technique we will also focus on a key idea, that our world is full of bacteria present virtually everywhere. Due to the ubiquity of microorganisms we will have to be extremely careful about contamination of our lab materials and the cultures we are working with. You will see that it takes very little exposure to introduce an unwanted organism to your materials.

The second part of the laboratory will continue our microscopic investigation into the microbial world by introducing you to the variety of morphologies (shapes) and cellular arrangements that are present among bacterial cells as well as comparing cell size. In addition, we will also focus on using the oil immersion lens which is a critical skill in the microbiology lab.

Cellular morphology and arrangement refers to the cell shape and the association shared between cells (if any). Although bacterial cellular morphology can be very diverse, there are three basic shapes of interest in our lab. They are coccus (pl. cocci), rod (sometimes called bacillus), and spiral. Based on how cocci cells divide they can designated as diplococci (pairs), streptococci (chains), tetrads (groups of 4), or staphylococci (clusters). Rod cellular arrangements can be described as singles, diplobaccilli (pairs), or streptobacilli (chains).

When comparing bacterial cell size, we will be using the metric system. The metric system is the standard system of measurement used in the sciences, including microbiology. The system makes measurement and unit conversions much simpler because all units and conversions are based upon the number 10.

Three of the most commonly measured properties are length, mass, and volume. The standard metric units for these variables are meter, gram, and liter. Prefixes are placed in front of each unit’s name to designate smaller and larger units. Let’s use length as an example to see how metric units work. One meter is equivalent to 39.37 inches, so it is roughly 1 yard in length. Now imagine dividing the meter into 10 equal-sized pieces. Each piece is 1 decimeter (dm) in length(1/10

th of a meter). Imagine dividing the meter into 100 equal-sized pieces. Each piece is 1

centimeter (cm) in length (1/100th

of a meter). Finally, divide the meter into 1000 pieces. Each piece is 1 millimeter (mm) in length (1/1000

th of a meter).

Bio 301 MICROBIOLOGY

Def an i that dise kno

Note the following:

1 m = 10 dm 1 m = 100 cm

1 m = 1000 mm1m = 1,000,000 µm

Most microorganisms are smaller than 1 mm, so we need to introduce an additional unit called the micrometer (µm). Imagine dividing 1 mm into 1000 equal-sized pieces. Each of these extremely small pieces is 1 micrometer in length. A typical bacteria cell is about 0.2-2.0 µm

in diameter and 2-8 µm in length.

Morphology/Arrangement Species Photo

Rods Singles Escherichia coli

streptobacilli Bacillus subtilis

TetradsMicrococcus luteus

staphylococciStaphylococcus aureus

Materials per pair:

3 TSA plates 2 sterile swabs

sterile waterprepared bacterial slides

Activity I: Ubiquity of MicroorganismsThe specific media type used in this lab is Trypticase Soy Agar (TSA) which is a complex mediaproviding a wide range of nutrients supporting a diversity of microorganisms.

37oC

U b i qu ity o f M i c r o o r g a n i s m s : S w a b Sa m p l e s 1. Obtain 3 TSA plates, flip the plates over, and label the

bottom with your group initials, lab day/time.2. Label one plate “air”. Remove the lid and leave it exposed

to the air until the end of the lab period.3. Add the following information on the remaining 2 plates:

a. Divide each plate into quadrantsb. Label one plate “37•C” and the other “25•C”.

inition of Fomite: nanimate object can transferase i.e. door b

25oC

c. Number each section 1-4, and make a key below of the four samples you would like to swab. You must use the same four sample areas for both plates

and these should include one fomite sample, two body samples, and one surface sample

Source 1: d i r t y f i n ge r Source 3:__________________Source 2: c l e a n f i n ger Source 4: _________________

BIOL1230 MICROBIOLOGY

4. In order to inoculate sources 1 and 2 start with dirty hands. Moisten your index finger with water then press your dirty index finger into quadrant 1 of both plates.

5. Now wash your hands following these directions from the Center for Disease Control:

a. Wet your hands with warm running water and apply soap.b. Rub hands together to make lather and scrub all surfaces.c. Continue rubbing hands for 20 seconds. (Imagine singing "Happy Birthday" twice)d. Rinse hands well under running watere. Dry your hands using a paper towel and use your paper towel to turn off the faucet.

6. Moisten your index finger again and press the clean index finger into quadrant 2 of both plates.

7. For sources 3 and 4: obtain two sterile swabs and one micro vial of water from the cart.

8. For source 3, moisten one swab and streak the area of interest thoroughly. Proceed to streak each of your TSA plates in the designated quadrant for that sample. B e s u r etor e m e m b e r ase ptict ec hniqu e ! (repeat for source 4)

9. Place the 37• C plate upside down (“bottoms up”) inside the incubator. Place the 25•

C plate upside down on top of the incubator. Since microbes are so small, it is necessary to allow time (24-48 hours) for them to multiply into populations so large we can see their colonies unaided. The plate incubated at room temperature will need a longer incubation (5-7 days).

We will be discussing throughout the semester basic requirements microbes have in order to live and replicate. These requirements include nutrition and environmental conditions such as temperature, pH, moisture, and oxygen. Microbes vary in their nutritional needs and their levels of tolerance to environmental conditions. Since both plates were streaked with the same samples and supplied the same nutritional media, it is interesting to expose each plate to a differentvariable – in this case temperature (either 25

oC or 37

oC). Both plates will be incubated in aerobic

atmospheric conditions.

Activity II: Cellular Morphology and Arrangement (prepared slides)The purpose of this second activity is 2-fold:

1) introduction to cellular morphology and arrangement2) proper use of the oil immersion (100x) objective lens.

1. Obtain 3 prepared slides representing different morphologies and arrangements.2. Begin focusing each slide on the 4x and then move to the 10x and 40x objective lenses. Once you have your image focused clearly on 40x move the turret in between the 40x and 100x objectives.

3. Without moving the stage or changing focus, drop a small, single drop of immersion oil onto the slide.

4. Move the oil immersion (100x) objective lens directly into the spot of oil.5. View slide—you might have to do some minimal focusing with the fine adjustment knob to clear up your image.

6. Sketch results in table provided.7. Clean oil off 100x object lens before moving to the next slide.


“AIR” PLATE—you should have left this plate uncovered on your lab bench the entire lab. At the end of the period be sure to close your dish and invert and incubate at 37

oC.

C lea n - u p a n d Dis po sal – f o ll o w i n st r u ct o r’ s d i r ecti on s. Once all items have been put in their assigned places wipe your work area with ethyl alcohol and wash your hands well.

E 2 R e s u l t s: ACTIVITY I RESULTS: Obtain your two sample plates you swabbed lastlab session and look for growth. Use the colony description figure takenfrom Science Buddies website

( h tt p :/ / www. sc i e n c eb u d d i e s . o r g / m e nt o r i n g / p r o j e ct _ i d ea s/ M i cr o Bi o _i m g _ 003 . gi f ) to assist you in describing bacterial growth for each quadrant. The results tables provided below are to be used to b ri e f l y describe what you observe. List each sample area in the space provided. The lower portion is for growth description. Include color, colony sizes, amount of growth, and colony description for each listed sample area.

Colony Description by Source for 37•C PlateSource 1: dirty index finger Source 2: clean index

fingerSource 3: Source 4:

Amount Description Amount Description Amount Description Amount Description


Colony Description by Source for 25•C PlateSource 1: dirty index finger

Source 2: clean indexfinger

Source 3: Source 4:

Amount Description Amount Description Amount Description Amount Description

“AIR” Plate Results: Did you have growth?ACTIVITY II RESULTS: For each slide sketch enough cells to demonstrate the morphology andarrangement of the bacteria viewed using the oil immersion (100x) objective lens.

Slide 1 Slide 2 Slide 3

SKETCH

Describemorphology andarrangement

E2 Write-upIntroduction paragraph: include a description of the following items from the backgroundinformation:• Ubiquity of microorganisms and aseptic technique• Discussion of culturing conditions provided including temperatures, media used, andatmospheric requirements• Description of different morphologies and arrangementsSubmit results from lab manualConclusion:• Use your data to support use of aseptic technique


• Use your data to address the use of optimal temperature during incubation• Discuss your observations of cellular morphology and arrangement


EXERCISE 3ASEPTIC TECHNIQUE AND MEDIA PREPARATION

Introduction

Asepsis means without contamination. The ability to carry out procedures without the

introduction of unwanted organisms, or contamination, is paramount to obtaining correctresults/identification. In addition, since some of these microbes are potential pathogens(organisms that cause disease), contamination could expose you and any other person you may come into contact with the possibility of infection. That is why vigilance in proper technique is necessary to reduce risk.Aseptic technique can be further divided into four categories including work area preparation,media preparation/handling, culture transfer, and clean-up and disposal.Work.

Work Area PreparationSince microbes are everywhere, even on us, it is necessary to begin to minimize the potential for contamination before we begin


to work with the microbes. Prior to beginning each laboratory session.


“AIR” Plate Results: Did you have growth?

ACTIVITY II RESULTS: For each slide sketch enough cells to demonstrate the morphology and arrangement of the bacteria viewed using the oil immersion (100x) objective lens.

assigned microscope #:

Slide 1 Slide 2 Slide 3

SKETCH

Describe morphology

and


• Ubiquity of microorganisms and aseptic technique• Discussion of culturing conditions provided including

temperatures, media used, and atmospheric requirements• Description of different morphologies and arrangements

Submit results from lab manualConclusion:

• Use your data to support use of aseptic technique• Use your data to address the use of optimal temperature during

incubation• Discuss your observations of cellular morphology and arrangement.



EXERCISE 3ASEPTIC TECHNIQUE AND MEDIA PREPARATION

IntroductionAsepsis means without contamination. The ability to carry out procedures without theintroduction of unwanted organisms, or contamination, is paramount to obtaining correctresults/identification. In addition, since some of these microbes are potential pathogens(organisms that cause disease), contamination could expose you and any other person you maycome into contact with the possibility of infection. That is why vigilance in proper technique isnecessary to reduce risk.Aseptic technique can be further divided into four categories including work area preparation,media preparation/handling, culture transfer, and clean-up and disposal.

Work Area PreparationSince microbes are everywhere, even on us, it is necessary to begin to minimize the potential forcontamination before we begin to work with the microbes. Prior to beginning each laboratorysession:

• Remove all you personal items away from the workbench except for your lab notebook and writing instrument.• Wipe down your workbench with ethyl alcohol (ETOH) and paper towels.• Wash your hands using CDC method as described in E2.• Begin gathering materials as instructed and outlined for that particular laboratory.

Media Preparation/HandlingWhen culturing bacteria you must provide them with all of the conditions they need for growthincluding nutrients, temperature, atmospheric requirements and more. Media is the nutrientmixture that is used to grow and keep microbes. Media is usually in broth or solid forms and willvary in content based on the goal of its use. These nutrient mixtures are normally in powderedform to increase their shelf life. When needed, the nutrient mix is added in a specific amount towater, boiled, transferred to containers, and autoclaved to sterilize. As long as the media remainsunopened in the original container, it should remain a sterile environment. If the media istransferred from the original container, care should be taken to avoid contamination. In the caseof the plastic Petri plates we will be using extensively in this lab section, media must betransferred. Plastic used for these plates are unable to withstand the temperature of the autoclavefor sterilization so they are shipped and remain in sterile packaging until their time of use. Theagar will be poured hot and quickly into the Petri dish to further avoid contamination.


Culture TransferAll of microbiology work involves transferring cultures to different growth media or onto slides. Itis critical when transferring bacteria that aseptic technique is maintained. A large portion oftoday’s lab procedure will emphasize how to move bacteria to different media types whilepreventing contamination.

Clean-up and Disposal –After EACH Lab (last step of aseptic technique)Unless otherwise instructed items used in the lab session should be treated in the following manner:Glassware - will be autoclaved and cleaned for re-use.

Test tubes/flasks –place in the designated test tube rack on the lab cart after the labeling has been removed. If an adhesive label is present, simply peel it off and place in the autoclave bag. If a marker has been used, wipe off with acetone prior to

Slides - If the slide has been heat fixed, wash with sponge and dish soap and place in designated container.

Disposable Items - items intended for one time use and items that cannot maintain their formand function after being autoclaved should be placed in the autoclave bag. These items will besterilized prior to their disposal to avoid contamination during and after waste collection. Theseitems include but are not limited to Petri plates and any paper or plastic items that have come into contact with microbes.

Goggles – clean with diluted dish soap, dry and then put away.

Once all items have been put in their assigned places wipe your work area with ETOH and wash your hands.

Activities Materials:4 test tubes of TSB hot pads 1 TSA plate flasks with filtered waterplates from E2 powdered mediastirrer/hotplate thermometersmagnetic stirrer

Aseptic TransfersA.) Sterile Broth to Sterile Broth:

A.) Sterile Broth to Sterile Broth:1. Obtain two sterile Trypticase Soy Broth (TSB) test tubes.

Label one tube “A” and one tube “B” and label both with your group initials and lab day/time.

2. Flame loop until the loop and some of the wire is red hot then let the loop cool.


3. Open test tube “A” using the pinky finger technique—hold the lid of the test tube with your pinky finger of your dominant hand, and use your other hand to twist the test tube away from the lid.

4. Flame the lip of the test tube

5. Get loopful of broth; make sure that only the sterilized portion of the loop makes contact with the broth.

6. Flame and close test tube.

7. Open test tube labeled “B” using the same technique as above.

8. Flame the lip of the test tube.

9. Inoculate test tube “B” with the loopful of sterile broth from tube “A”. Be sure that only the sterilized portion of the loop makes contact with the broth.

10. Flame and close test tube “B”.11. Flame loop red hot.12. Place both test tubes in a rack in the incubator.

B.) Sterile Broth to Sterile Plate:

B.) Sterile Broth to Sterile Plate:

1. Obtain one sterile TSB test tube and a sterile Trypticase Soy Agar plate (TSA). Label both with your group initials and lab day/time. Label the test tube “C” and the plate “D”. Be sure to put your labels on the bottom of the Petri dish.

2. Flame loop until the loop and some of the wire is red hot then let the loop cool.

3. Open the test tube using the pinky finger technique (see above)


5. Get loopful of broth; make sure that only the sterilized portion of the loop makes contact with the broth.


7. Open the Petri dish just enough to get the loop in. 8. Very gently move the loop across the Petri dish. Be careful not to dig into or make any

breaks in the agar bottom.

9. Close the Petri dish.

10. Flame loop red hot.


11. Invert the Petri dish and add it to your class’ stack in the incubator. Put the test tube in the incubator rack too.

C.) Colony to Broth Transfer

1. Obtain one sterile TSB test tube and a plate with growth from E2. Label with your group initials, lab day/time and “E” on the TSB tube.

2. Flame loop until the loop and some of the wire is red hot. Allow loop to cool.

3. Open the Petri dish just enough to get the loop in and very gently use your loop to pick up an isolated colony of bacteria. Be sure not to disturb the agar or any neighboring

colonies.

4. Close the Petri dish.

5. Open the test tube using the pinky finger technique (see above).


7. Inoculate the broth; make sure that only the sterilized portion of the loop makes contact with the broth.


9. Flame loop red hot.

10. Put the test tube in the incubator rack.


Media PreparationEach group will be asked to prepare media. The type of media and the specific instructions will beassigned by the instructor. It will take just a few minutes to get the media measured and added tothe flask. It will take about 20-30 minutes for the media to reach boiling before it is ready to pourinto test tubes. Instructors will need to make sure that each class’ tubes are autoclaved as soon as possible and then stored in the refrigerator as room allows. The basic procedure to be usedby all classes is as follows:

1. Measure out the proper amount of media powder for ½ L of media using the electronicbalance.

2. Fill up your flask with the proper volume of filtered water (1/2 L) and place on thestirrer/hotplate. Drop in a magnetic stirrer and turn the stirrer on 6-8.

3. Slowly pour in the media powder into the water.

4. Turn the hotplate on high. Agar can superheat so it is important to keep an eye on yourboiling agar. Also agar will not dissolve in water that is hot, so DON’T heat the water untilafter adding the powder.

5. Once the agar or broth has reached boiling point (use thermometers to register 100oC). Youwill notice that the liquid is clear rather than cloudy indicating that the media has dissolved.Also there is often a frothy head that forms on the top of the boiling media (when TSA andTSB start to look like beer—they are ready).

6. Follow instructor’s directions on how to pour the media into the test tubes given. Loosely capthe test tubes and put autoclave indicator tape on the rack. Be sure to provide a label on therack with the type of media, date made, and group initials. Your instructor will help you getyour rack autoclaved with the rest of the class’ tubes.


7. Test tubes need to be autoclaved as soon as possible after being poured. Once sterilizedtest tubes can be stored in the refrigerator.

E3 ResultsFor each item record whether or not you had growth. If you have growth in TSB then the testtube will be cloudy. You might need to vortex your TSB to see growth as the bacteria can settle.

Procedure Materials Growth? Yes or No

Sterile TSB -7 Sterile TSB Tube “A”

Tube “B”

Sterile TSB -7 Sterile TSA Tube “C”

TSA plate “D”

Isolated colony -7 sterile TSB Tube “E”


1. Aseptic technique define, state purpose, give some examples ofSubmit results from lab manualConclusion:

2. Did your results turn out as expected? Explain.3. Describe some procedural errors that could negatively affect your results.


.

EXERCISE 4DIFFERENTIAL STAINING TECHNIQUES – GRAM STAIN AND SPECIAL

STRUCTURE STAINS

Introduction

As we have seen in the previous exercises, light microscopy and the use of a stain are valuable tools for viewing bacteria. This allows us to see the morphology of a microbe of interest. A differential stain technique allows additional discrimination and helps to narrow down the list of possibilities with regard to its identity. A differential stain would be one that allows determinationof differences between species having similar morphologies based on their ability to take the stain.

In multiple stain procedures, the initial stain is retained by cells that are termed “positive ”while the “negative”cell loose the initial stain. This necessitates the addition of a secondary or counter stain, to make the negative cells visible under the microscope. Differential stains show a difference in chemical composition, metabolism, and/or the presence of a special structure.


The first step in making a slide is to smear the bacteria onto the slide. Preparation procedures forthe smear will vary according to cell concentration in the culture. Generally a broth culture will beless concentrated than surface cultures and can be smeared directly onto the surface of a cleanslide and allowed to air dry. Surface cultures (ones growing on agar) need to be diluted in order todiscriminate single cell shapes once stained. A loop of water is added to the slide and the loop of sample is mixed in and distributed on the surface of the slide and allowed to dry.

The second step in making most differential stains is to heat fix the slide. A heat fixed slide isone where the organisms are placed on the slide and the slide is allowed to dry. After the slide isfree of visible moisture, it is passed through an open flame 2-3 times before the staining begins. Itis imperative that the slide is completely dry before the heat-fix step; otherwise the bacterialproteins can be denatured during the heat-fix step and cause a distortion in the cell morphology.The purpose of the heat-fix process is to affix them to the surface of the slide so that they are notwashed off during the staining process. When done correctly, heat-fixing will also kill theorganisms.

The most well-known and used differential stain is the Gram stain. This stain processdetermines differences in peptidoglycan (a chemical found only in bacteria) on the outside of thecell wall. Bacteria can be divided into one of two categories based on their gram reaction, either gram positive or gram negative. Gram positive cells have a thicker peptidoglycan cell wall than gram-negative cells, and retain the initial stain, crystal violet (purple), in the gram stain procedure. Gram negative cells are decolorized during the gram stain procedure which requires the application of a counter stain, Safranin (pink), so that these cells can be easily viewed.

The acid-fast stain is used in a limited number of cases to stain organisms that have an additionof mycolic acid in their cell walls which prevents them from Gram staining. The Mycobacteriumspecies require this type of stain process in order to view them. Due to the nature of the waxymycolic acid, these cells must first be steamed in order for the stain to penetrate the cell wall. In this procedure acid-fast cells retain the initial stain, carbolfuchsin (reddish-purple), while negativeCells are stained by the secondary stain, Methylene blue (blue).

Some species have cellular structures that are uncommon and can be used in identification.Endospores are examples of specialized cellular structures produced by some Bacillus andClostridium species in response to stressful environmental conditions. Endospores are dormantcells able to survive hazardous conditions that later germinate to form vegetative (metabolically


active) cells when conditions improve. The endospore stain employs a multiple stainprocedure that stains both endospores and vegetative cells. The endospore stain involves steamsimilar to the acid-fast stain. Without steam the tough spore coat prevents the endospore fromtaking up stain. During this procedure, endospores keep the initial stain, Malachite green (green),while the vegetative cells retain the secondary stain, Safranin (pink).

Other important structures that can be detected with stains are flagella. These structures areResponsible for the motility of an organism. While the presence of flagella alone can be importantin characterizing bacteria, the ability to describe the arrangement of the flagella can be even moreuseful. For direct observation of flagella a tricky staining procedure is used. Motility media is analternative method to staining that allows for indirect observation of flagella. Due to the dilutenature of this media microbes are able to travel in the media if they are mobile. This takes timeAnd incubation will be necessary. In addition the media has a chemical added, TCC, whichmicrobes take into their cells. When metabolized, the TCC changes from being colorless to a pink color allowing easier viewing of the placement of the growth.

There are plenty of resources on the internet that can help you visualize these processes a little better. Check out the following links:

• Gram Stain: http://faculty.mc3.edu/jearl/ML/mL-5.htm• Acid-fast Stain: http://faculty.mc3.edu/jearl/ML/mL-6.htm• Endospore stain: http://www.slic2.wsu.edu:82/hurlbert/micro101/pages/101lab6.htmL

A c ti v i t y M a t er i a l s : Per team K. pneumoniaPer Class: M. smegmatis (pathogenic) S. epidermidis Blank slides B. subtilis P. aeruginosa Hotplates/beakers E. coli 3 motility media test tubes

Students will have 2 lab periods to complete E4.

• Each group should start by inoculating the 3 motility tubes1. Obtain 3 test tubes of motility media and label “1-3”, group initials, and class period.

1. Escherichia coli2. Pseudomonas aeruginosa3. Klebsiella pneumoniae

2. Using aseptic technique, stick the sterile inoculation needle into the broth culture (be sure to flame the entire length of the needle).3. Insert the needle straight down into the motility media and pull the needle straight back out.4. Fire the tube and quickly replace the lid. Fire the needle5. Place in the test tube rack in the incubator for 48 hours.


• Each student should obtain a total of 8 slides and label slides “a-h” (see List of bacteria below). Make all 8 slides using the heat-fix procedure. After Heat-fixing all 8 slides begin staining slides starting with the 4 gram stain slides.

Slide label Organism Stain Technique“a” and “f” Staphylococcus epidermidis Gram stain and acid-fast

stain“b” Pseudomonas aeruginosa Gram stain“c” and “h” Escherichia coli Gram stain and endospore

stain“d” and “g” Bacillus subtilis Gram stain and endospore

stain“e” Mycobacterium smegmatis

(PATHOGENIC)Acid-fast stain

“i” MIXED slide of S. epidermidis and E. coli Gram stain

For All slides—make smear using heat-fix procedure:

From Solid Sample: From Liquid Sample:1. Clean and label bottom of slide 1. Clean and label bottom of slide2. Add loopful of water 2. Flame loop red hot3. Flame loop red hot 3. Vortex Sample4. Aseptically obtain bacteria (isolated 4. Flame test tube openingcolony if possible) 5. Aseptically obtain bacteria5. Smear (spread-out) bacteria on 6. Flame test tube openingSlide 7. Smear (spread-out) bacteria on slide6. Flame loop red hot 8. Flame loop red hot

7. AIR DRY COMPLETELY 9. AIR DRY COMPLETELY8. Heat-fix (pass slide through flame) 10. Heat-fix (pass slide through flame)9. Proceed to directions below for the 11. Proceed to directions below for the specific differential stain. specific differential stain.

Under oil immersion, observe your slides--remember you are looking for differences inmorphology, size and arrangement in addition to whether the organism is positive or negative forthe differential stain. Then sketch your observation in the results section. (Don’t forget to cleanyour microscope BEFORE & AFTER use.)


Procedure I

Gram Stain ProcedureOrganisms:a. Staphylococcus epidermidisb. Pseudomonas aeruginosac. Escherichia colid. Bacillus subtilisi. MIXED slide of S. epidermidis and E. coli

1. Prepare slides for Gram stain as instructed above.2. Attach a clothespin to the end of the slide and hold it over the sink.3. Cover the slide with Crystal Violet stain and allow it to react for 1 minute4. Rinse the slide with a small amount of running water and remove the excess water by gently shaking the slide.5. Cover the slide with Gram’s Iodine--allow it to react for 1 minute.6. Rinse the slide with water as in Step 4.7. Hold the slide at an angle and apply 1 drop of decolorizer8. Immediately rinse the slide thoroughly and remove any excess water as in Step 4.8. Immediately rinse the slide thoroughly and remove any excess water as in Step 4.9. Cover the slide with Safranin for 1 minute.10. Rinse the slide thoroughly.11. Blot the slide dry by placing it in between the sheets of the bibulous paper and lightly pat with your hand.

Apply immersion oil when ready to view & record in Results section

Procedure II

Acid-fast Stain ProcedureOrganisms:e. Mycobacterium smegmatis (pathogenic)f. Staphylococcus epidermidis

1. Prepare slides as instructed above.2. Cover the slides with a piece of paper towel the same size As the smear.3. Place clothespins at both ends of the slide to form a rack And place it on top of the steaming water beaker.4. Flood the slide with Carbolfuchsin stain.5. Gently steam the slide for 10 minutes, reapplying the stain as needed to prevent the slide from drying out.6. Remove the paper towel carefully with forceps and place in trash.7. Rinse the slide with a small amount of running water until the excess stain is removed.8. Hold the slide at an angle and apply acid alcohol by making drops


at the highest part of the slide (Near the clothespin “handle”) and allow it to drip down the slide. Do this for 25-30 seconds.9. Rinse the slide.10. Apply several drops of Methylene Blue stain and leave for 45 seconds.11. Rinse the slide thoroughly and blot dry.


Procedure III

Endospore Stain ProcedureOrganisms:g. Bacillus subtilish. Eshericia coli

1. Prepare slides as instructed above.2. Apply clothespins to each end of the slide and place over a steaming beaker of water (like acid-fast).3. Apply a piece of paper towel cut to the size of the slide on the surface of the slide.4. Flood the paper towel with Malachite Green stain.5. Allow the slide to steam for 5 minute and reapply the stain as needed to prevent the paper Towel from dying out. 6. Remove the paper towel gently using forceps and remove one clothespin.7. Rinse the slide with a small amount of running water.8. Hold the slide over the sink and apply Safranin and allow it to react for 1 minute.9. Rinse and blot dry.



E4 Write-upIntroduction paragraph: include a description of the following items from the backgroundInformation:• Gram stain, endospore stain,acid-fast and motility mediaSubmit results from lab manualConclusion:• Use your results to describe the cellular characteristics of each organism testedThis write-up must be typed and be in your own words.

E4 ResultsProcedure I-Gram Stain assigned microscope #:_____________________

Slide Label Organism Sketch & Indicate Colora. Staphylococcus epidermidisb. Pseudomonas aeruginosac. Escherichia colid. Bacillus subtilisi. MIXED S. epidermidis and E.

coliProcedure II-Acid-fast assigned microscope #:_____________________

Slide Label Organism Sketch

e. Mycobacterium smegmatis(pathogenic) (pathogenic)

f. Staphylococcus epidermidis

Procedure III-Endospore Stain assigned microscope #:_____________________

Slide Label Organism Sketch & Color


g. Bacillus subtilis

h. Escherichia coli

Procedure IV-Motility Results: Draw the regions where growth was observed.

Organism 1.) Escherichia coli 2.) Pseudomonas aeruginosa

2.) PseudomonasAeruginosa

Sketch

See previous page for E4 write-up assignment.

EXERCISE 5CULTURING: MEDIA SELECTION AND INOCULATION

TECHNIQUES

Introduction:In the previous four exercises we became familiar with visual techniques used in microbiology.You learned to describe various colony characteristics that, although not often, may aid in thedetermination of a particular microbe. We also examined various types of bacteria under themicroscope to record cell morphology and any biochemical/structural differences that could beobtained through the use of staining procedures. Although helpful, determination of cell type andstructure is limited when it comes to correctly identifying a microorganism. There are manyspecies that, for example, are Gram negative rods with motility. When studying a bacterium orwhen trying to diagnose a disease for proper treatment, the exact species (or even strain of thatspecies) must first be determined.


For further characterization we look to various metabolic differences that are specific to that typeof bacteria. All living organisms must acquire energy from their environment. There aredifferences in energy sources and metabolic pathways between groups of bacteria. There arealso ranges of environmental conditions that are particular to groups of bacteria (pH, temperature,atmospheric requirements, etc.) where they best function (refer to E2 for temperature differences).Knowing and manipulating these conditions provide excellent tools for correctly identifyingbacteria. The next two exercises will focus on various procedures that have been developed tomanipulate differences in energy needs and environmental condition ranges.

Exercise 5 will be focusing on the use of media. For solid media, agar base (solidifying agent) isadded to a broth containing nutrients that provide energy for microbes so that they maymetabolize and replicate. The ingredients that are present in the broth will determine what groupsof microbes are able to metabolize and grow. There are some metabolic differences amongsimilar microbes that can be used for identification (see differential media below). Metabolicdifferences are often observed using colorimetric tests which typically incorporate either a pHindicator that will change the color of the media if there is a change in pH or a chemical thatattaches to the metabolite of interest and concentrates in the microbes changing the normalcolony color.

In general there are three different groups of media type used in this lab:• Complex media –is comprised of partially digested chemical compounds from organic substances such as yeast, meats, dairy products, tissues, or vegetable materials. The amount of each cannot be known due to differences between the organic compounds and the amount of digestion that has occurred. This knowledge is not necessary when culturing most types of microbes. This media type is useful when trying to grow out bacteria, or when culturing a mixed diverse sample (see E2). An example of a complex media is Trypticase Soy Agar (TSA).

• Selective media – is used to select, or isolate, specific groups of bacteria. This is usually based on a known environmental condition range they can tolerate that most groups cannot.

• Differential –provides chemical compounds that bacteria metabolize differently. This difference is observed by colony or media color change once the microbe has been introduced to the media and allowed to grow and metabolize. Often this type of media is used to differentiate between similar groups of organisms with the same morphology and biochemical resemblances.

There are several medias that are both selective and differential these include EosinMethylene Blue and Mannitol Salt Agar.

Eosin Methylene Blue (EMB) is used to differentiate between gram-negative,enteric, rods because this media primarily supports (or selects for) the growth ofthese organisms. Growth on EMB can be used to differentiate organisms basedon their ability to ferment lactose. During the fermentation process, acid isproduced as a waste product. When grown on EMB this acid production results


in a colony color change ranging from pink to purple. In the case of E. coli andKlebsiella pneumoniae, colonies can appear dark purple with a metallic greensheen. One exception to EMB selectively growing gram-negative rods is thegram-positive cocci Enterococcus faecalis. E. faecalis is able to grow on this

media because it is an enteric bacterium.

Mannitol Salt Agar (MSA) contains salt that most organisms cannot tolerate due to theirosmolarity ranges. Microbes that normally exist in anenvironment with this condition, such as microbes of the skin, are able to grow. For those halotolerant organisms, MSA is alsoused to differentiate species based on their ability to fermentmannitol. Organisms that can ferment mannitol produce an acidby-product causing the pH indicator in the media to turn from pinkto yellow.

In addition to using color changes to differentiate bacteria,location of growth in a special media can be useful as we have already seen with motility media(E4). Media can be used to determine whether a microbe is an obligateaerobe (A), obligate anaerobe (B), or a facultative anaerobe (C) speciesby observing location of growth in special media (see figure). An oxygengradient is formed in the oxygen requirement media once the test tube isboiled. An aerobic environment remains in the top 1-2 cm of the test tube,while anaerobic conditions are present below this point.

When working with media it is important that you place thebacteria on the media in an informative way. When tryingto simply grow bacteria on a media the best method touse is a streak-to-grow technique. With this methodyou are simply trying to get the bacteria on the media and you can either use aback-and-forth motion on the plate with your loop or one distinct streak. If you wantto obtain isolated colonies for use in working with a pure culture, than a T-streak

method would be more appropriate. The idea is to dilute or disperse the cells sowhen incubated, they will form isolated colonies that are separated to such a degreethe colonies do not touch. This will allow viewing of the individual colonies for anydistinguishing culture characteristics as well as for creating and working with a pure culture.

ActivitiesMaterialsPer team:4 TSA plates2 EMB plate2 MSA plates4 Oxygen Requirement tubes


Per table:Test tube rack containing Escherichia coli, Staphylococcus epidermidis, Staphylococcus aureus,Pseudomonas aeruginosa, Enterobacter aerogenes, Enterococcus faecalis, Klebsiellapneumoniae, Clostridium perfringens, and Micrococcus luteusActivity I: Inoculation and Isolation of ColoniesIsolation of a Colony: T-Streak Technique

1. Obtain a TSA plate and label on the back your group’s initials, YOUR NAME, classtime, “1-4”, and divide your plate by drawing a T (see below).

1. Staphylococcus epidermidis2. Pseudomonas aeruginosa3. Escherichia coli4. Enterobacter aerogenes

2. Sterilize your transfer loop and allow it to cool.3. Aseptically obtain a loop of broth from one of the cultures (see box below).4. Streak the top section with closely spaced streaks that DO NOT overlap5. FLAME loop6. Drag bacteria from top section into right hand section, streak as in step 47. FLAME loop8. Drag bacteria from right section to left section, streak as in step 49. FLAME loop10. Invert and Incubate at 37oC


Activity II: Media Selection (MSA & EMB)

1. For each type of media plate (2 MSA & 2 EMB) label with your initials, class day/time, and label one set of MSA and EMB plates as “a-d” (see figure) and the second set of plates “e-h”.2. For each plate aseptically streak to grow with the appropriate culture. Remember toalways practice good aseptic technique, especially between the transfers of the differentspecies.

a. Staphylococcus epidermidisb. Staphylococcus aureusc. Micrococcus luteusd. Enterococcus faecalis

e. Pseudomonas aeruginosaf. Escherichia colig. Enterobacter aerogenes.h. Klebsiella pneumoniae

3. Invert and incubate all 4 plates 37oC.


Activity III using media to determine O 2 requirements:

1. Obtain 4 Oxygen Requirement test tubes. Label with initials, day/time, and “5-8”.

2. Allow the oxygen requirement tube to cool to body temperature (it will feel comfortablywarm to the touch). DO NOT allow your media to solidify before inoculation. Be sure tosterilize the full length of the loop and wire to prevent cross contamination of the tubes.

3. Inoculate the test tube with the appropriate microbe. To do this, put the loop withinoculate straight down to the bottom of the tube and pull out. Repeat for all 4 species.

1. Klebsiella pneumoniae2. Pseudomonas aeruginosa3. Escherichia coli4. Clostridium perfringens

E5 Results:Activity I: Inoculation and Isolation of Colonies Obtain your best T-Streak plate and observe the growth. Sketch yourresults in the diagram below, and then count the total number of isolatedcolonies you observe.Total Isolated colony count: _____

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