Exploring the Infectious Nature of Viruses

Transcription

1 Edvo-Kit #209 Exploring the Infectious Nature of Viruses Experiment Objective: This experiment explores the life cycle of viruses using classroom-safe bacterial viruses. Students will use the viral plaque assay to visualize the infection of E.coli by Bacteriophage T4 and determine the viral titer. See page 3 for storage instructions. SAMPLE LITERATURE 209 Please refer to included weblink for correct version

2 EXPLORING THE INFECTIOUS NATURE OF VIRUSES EDVO-Kit 209 Table of Contents Page Experiment Components 3 Experiment Requirements 3 Background Information 4 Experiment Procedures Experiment Overview 8 Using the Plaque Assay to Determine Viral Titer 9 Experimental Results and Analysis 11 Study Questions 12 Instructor's Guidelines 13 Pre-Lab Preparations 14 Experiment Results and Analysis 16 Study Questions and Answers 17 Appendices 18 Safety Data Sheets can be found on our website: EDVOTEK and The Biotechnology Education Company are registered trademarks of EDVOTEK, Inc. ReadyPour, COLORTOP, BactoBeads, and ViroBeads are trademarks of EDVOTEK, Inc. 2

3 EDVO-Kit 209 EXPLORING THE INFECTIOUS NATURE OF VIRUSES Experiment Components REAGENTS & SUPPLIES Storage Check ( ) E.coli BactoBeads 4 C with dessicant Bacteriophage T4 ViroBeads 4 C with dessicant ReadyPour Luria Broth Agar Room Temp. COLORTOP Agar Room Temp. 1X Phosphate Buffered Saline (PBS) Room Temp. Luria Broth Room Temp. 15 ml screw-cap conical tubes Room Temp. 1.5 ml snap-top microcentrifuge tubes Room Temp. Sterile loops Room Temp. Petri Plates (large) Room Temp. Wrapped 10 ml pipet (sterile) Room Temp. Experiment #209 is designed for 10 lab groups. Requirements (not included with this kit) 37 C incubation oven Waterbath Automatic micropipets (5-50 μl, μl) and tips Pipet pump White light box (optional) All experiment components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor administered to or consumed by humans or animals. 3

4 EXPLORING THE INFECTIOUS NATURE OF VIRUSES EDVO-Kit 209 Background Information WHAT IS A VIRUS? Table 1: Viruses that Affect Human Health In the late 1800 s, scientists were working to characterize Class Genome Envelope? Example of Disease the means through which microbes could cause Adenovirus dsdna No Respiratory Infection disease. While some diseases like cholera, tuberculosis Herpesvirus and anthrax could be explained by the presence of dsdna Yes Chicken Pox, Shingles specific bacteria, other diseases, like rabies, could not. As such, these agents were considered to be a biological Parvovirus Reovirus ssdna dsrna No No Rashes Diarrhea chemical ; accordingly, they were named virus, Flavivirus ssrna Yes West Nile Virus from the Latin word that refers to poison or other venomous compounds. At this time, scientist Martinus Filovirus ssrna Yes Hemorrhagic fever Beijerinck was studying the infectious agent that caused tobacco mosaic disease, a plant disease that causes widespread crop damage. Through his studies, Retrovirus ssrna Yes Human Immunodeficiency Virus Beijerinck proposed that the virus that caused this disease must be much smaller than bacteria, as it could not be visualized by light microscopy. Furthermore, since viruses could not be cultured in nutrient media, they must only be able to replicate in their host organism. The theory that viruses were in fact infectious particles was confirmed in 1935, when Wendell Stanley visualized the tobacco mosaic virus using an electron microscope. Today, virology is a specialization of microbiology that explores viruses and the diseases that they cause. Studies have shown that viruses are very simple infectious agents, comprising little more than a DNA or RNA genome surrounded by a protective protein coat. As such, viruses rely entirely on a host organism for basic biological functions, including DNA replication, transcription, and basic metabolism. For example, viruses lack ribosomes, which are responsible for protein synthesis. Therefore, for viral RNA to be translated into protein, it must take over the translation machinery of its host organism. Early experiments characterizing the life cycle of viruses contributed to early discoveries in molecular biology, lending valuable insights to the processes of DNA replication, transcription, and translation. Capsid Helical (Tobacco Mosaic Virus) Icosahedral (Bacilliform Virus) Complex virus (T4 Bacteriophage) Furthermore, virology addresses public health concerns since viral infections impact the health of their host (Table 1). Some viral infections, like the common cold, are relatively harmless. The majority of cold symptoms (fatigue, fever and body aches) result from the body s innate response to the infection, not the virus itself. Other viruses, like Human Papillomavirus or Hepatitis B, integrate their genes into the host genome. The proteins coded for by these genes will influence the behavior of the host cell, transforming a normal cell into a cancer cell. Moreover, the effects of emerging viruses like Ebola or certain strains of Influenza on their human host can be deadly. Prolate (Adenovirus) Envelope (HIV virus) Figure 1: Structure of Viral Capsids 4

5 EDVO-Kit 209 EXPLORING THE INFECTIOUS NATURE OF VIRUSES Viruses are classified by the nucleic acid composition of their genome and by whether the nucleic acid is singlestranded or double-stranded. A special layer of protein called the capsid surrounds and protects the viral genome (summarized in Figure 1). This protective shell is built from protein subunits called capsomeres that assemble into three major structural forms: helical, icosahedral and prolate. Some capsids are further surrounded by a lipid bilayer envelope, which assists with viral entry into the host cell. Other viruses display a combination of two or more structural forms and are thus categorized as complex. Viral capsids range in size from five to three hundred nanometers in diameter, making it impossible to visualize most viruses by light microscopy. Viral Replication Takes Place in a Host Organism Viruses are not considered to be free-living organisms all viruses require a host organism to replicate. For example, the viruses known as bacteriophages infect and replicate within bacteria. A typical bacteriophage comprises an elongated capsid head that encloses the DNA and a protein tailpiece that facilitates attachment of the virus to the bacterial host. After the bacteriophage injects its DNA into its host, the virus replicates using one of two different methods: the Lytic Cycle or the Lysogenic Cycle. In the Lytic Cycle, the bacteriophage takes over the cell s metabolic machinery to reproduce. After building new phage particles, viral enzymes cause the bacterial cell to break down, or lyse, releasing the virus particles. The life cycle of a lytic phage like T4 phage progresses through a series of five steps: Attachment, Penetration, Replication, Assembly, and Release (summarized in Figure 2). Attachment occurs when the T4 phage binds to specific receptors on the surface of the E. coli bacterium. The phage then penetrates through the bacterial cell wall and injects its genome, leaving the phage coat behind. At this time, viral enzymes break down the host DNA. The phage recycles T4 Phage 1. Attachment the nucleotides from the host DNA to replicate its own DNA. The phage also uses the host cell machinery to produce important proteins, including components of its protein coat. These newly synthesized capsomeres self-assemble around the viral genome, resulting in new viruses. 5. Release Viral DNA released 2. Penetration The last step of the viral reproduction cycle is the release of the new phage viruses into the surrounding environment. Release depends on the production of the viral protein holin, which creates pores in the bacterial cell membrane, and lysin, which degrades the bacterial cell wall. This damage encourages extracellular 4. Assembly Figure 2 - The Lytic Cycle of T4 Phage 3. Replication 5

6 EXPLORING THE INFECTIOUS NATURE OF VIRUSES EDVO-Kit 209 fluid to enter the bacterium, swelling and eventually bursting the cell. Once the bacterial cell lyses, the new viruses can infect nearby bacterial cells. The life cycle of bacteriophage T4, from penetration of the phage to the release of new phage particles, takes about 40 minutes (Figure 3). In contrast to the lytic cycle, viruses that 0.1 replicate via the Lysogenic Cycle survive in Eclipse the host cell without causing any immediate harm. Instead, temperate phages like Latent period Rise period bacteriophage lambda (or phage λ) integrate their genome into that of the host Figure 3 - Timeline of Phage Infection cell. Similar to the lytic cycle, the lysogenic cycle begins when the phage attaches to the host cell and injects its DNA. Instead of immediately forcing the host cell to produce new virus particles, the injected viral DNA integrates with the bacterium s chromosome. Each time the host cell divides, the latent viral DNA (known as a prophage) is also copied and passed into the daughter cell. The viral genome exists in a quiescent state until the cell is exposed to adverse conditions such as exposure to UV or mutagenic chemicals. At that time, the viral genome excises itself from the host cell chromosome and the lytic cycle is initiated (Figure 4). Virus Quantification Using the Plaque Assay Viral quantification techniques are performed to determine the number of viruses in a given sample. As such, this is an essential technique in the academic, industrial and medical sectors. For example, virologists in academic labs study the effects of the viral infections on the host organism. The multiplicity of infection (MOI) is calculated before performing the experiment to ensure that enough virus is added to the experiment to infect the entire sample. In industry, viruses are used Bacteriophage per infected bacterium Virus attaches to cell wall and injects viral DNA. Viral DNA forms a circle. Under Adverse Conditions LYTIC CYCLE New viral DNA and proteins are produced & assembled. Viruses are released. Under Normal Conditions LYSOGENIC CYCLE Viral DNA inserts itself into the bacterial chromosome. Total phage Figure 4 - The Lysogenic Cycle of T4 Phage Extracellular phage Occasionally, the viral DNA separates from the bacterial chromosome, initiating a Lytic cycle. Time (minutes) Cell and viral DNA are reproduced many times. Average burst size Bacterium reproduces normally, replicating viral DNA at each cell division. 6

7 EDVO-Kit 209 EXPLORING THE INFECTIOUS NATURE OF VIRUSES to introduce recombinant DNA into mammalian cells to produce biological medications like the hemophilia drug Factor VIII. This method of gene transfer, called transduction, creates cell lines with the foreign DNA permanently integrated into the host cell s genome. After purifying the medication, the pharmaceutical company must ensure that it is free from viral contamination. Furthermore, clinicians will often determine a patient s viral load, or the amount of virus in a patient s sample, to determine the severity of the infection. For example, the viral load of HIV patients is regularly monitored to ensure that the treatment is effective. 10 µl 100 µl 100 µl 100 µl 100 µl 100 µl 990µl 900µl Serial Dilution Virus Stock Adsorb virus dilutions to cell monolayer. Agarose overlay Plaques develop in the cell monolayer Figure 5 - Plaque Assay Fix and stain the cells, count the plaques If a virus is lytic, virologists may quantify the number of infectious particles present in a sample using the plaque assay (outlined in Figure 5). A monolayer of host cells (prokaryotic or eukaryotic) is plated on a solid nutrient medium and infected with the virus. The samples are incubated for a period of time, during which the virus begins to reproduce within its host. Eventually the host cell will lyse, releasing the virus from the infected cell into the environment, where they will infect the surrounding cells. This creates a visible region of clearing called a plaque. The plaques are counted to calculate the initial concentration of virus particles present within the sample. Each dilution is performed in triplicates to minimize error and to obtain a more accurate concentration. In this experiment, we will use the T4 phage and its host organism E. coli to study the dynamics of a viral infection. The assay is performed using double agar overlay method, which creates a thin, homogenous layer of the bacteria/phage mixture in soft agar on top of a hard agar base. By trapping the microbes in a layer of agar, the virus is only able to spread to cells in its immediate vicinity. The soft overlay is prepared by melting the agar at 95 C, then cooling it to 60 C. At this temperature, the agar is warm enough to remain liquid, but cool enough to add the E.coli/phage suspension without killing the bacteria. The agar and bacterial suspension are mixed and evenly poured over a hard agar base plate. Once the agar has solidified, the plates are incubated for 24 hours, during which time the bacteria and the virus grow. In order to determine the concentration of the virus, or the titer, serial dilutions of the viral stock sample are prepared. Bacterial samples are inoculated with a small volume of each dilution, and assayed using the plaque assay. The concentration of a bacteriophage stock is determined in plaque-forming units (PFU) per milliliter. To calculate the virus concentration, the plaques formed on the agar plate are counted and multiplied by the dilution factor. For a valid phage count, the number of plaques per plate should be between 30 and

8 EXPLORING THE INFECTIOUS NATURE OF VIRUSES EDVO-Kit 209 Experiment Overview EXPERIMENT OBJECTIVE: This experiment explores the life cycle of viruses using classroom-safe bacterial viruses. Students will use the viral plaque assay to visualize the infection of E.coli by Bacteriophage T4 and determine the viral titer. LABORATORY SAFETY 1. Gloves and goggles should be worn routinely as good laboratory practice. 2. Exercise extreme caution when working with equipment that is used in conjunction with the heating and/or melting of reagents. 3. DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS. Wear gloves and safety goggles 4. Exercise caution when using any electrical equipment in the laboratory. 5. Always wash hands thoroughly with soap and water after handling reagents or biological materials in the laboratory. LABORATORY NOTEBOOKS: Scientists document everything that happens during an experiment, including experimental conditions, thoughts and observations while conducting the experiment, and, of course, any data collected. Today, you ll be documenting your experiment in a laboratory notebook or on a separate worksheet. Before starting the Experiment: Carefully read the introduction and the protocol. Use this information to form a hypothesis for this experiment. Predict the results of your experiment. During the Experiment: Record your observations. After the Experiment: Interpret the results does your data support or contradict your hypothesis? If you repeated this experiment, what would you change? Revise your hypothesis to reflect this change. 8

9 EDVO-Kit 209 EXPLORING THE INFECTIOUS NATURE OF VIRUSES Using the Plaque Assay to Determine Viral Titer Serial Dilution 180µl PBS 180µl PBS 20µl 20µl 20µl 180µl PBS 1:10 1:100 1: µl of virus solution µl PBS 20 µl of 1:10 virus solution µl PBS 20 µl of 1:100 virus solution µl PBS Virus solution 1:10 1:100 1:1000 Table 2: Making Serial Dilutions Label Plates Control 1:10 1:100 1: LABEL four 1.5 ml microcentrifuge tubes as follows: 1:10, 1:100, and 1: ADD 180 μl of PBS to each of the labeled microcentrifuge tubes. 3. PREPARE serial dilutions of the virus solution using Table 2. For example, to prepare the 1:10 dilution, add 20 μl of the virus solution to the tube labeled 1:10 containing 180 μl PBS. 4. LABEL five agar base plates as follows: CONTROL, 1:10, 1:100, and 1:1000. Wear gloves and safety goggles 100µl Bacterial growth Roll 2-3 sec Quickly Pour Circular motion WARNING! Bacteria and Phage must be added, mixed, and poured quickly to prevent hardening of the COLORTOP Agar. COLORTOP Control Tube Control 5. PREPARE the control sample. ADD 100 μl of bacterial growth to the COLORTOP Agar tube labeled CONTROL. 6. ROLL agar tube between your palms for 2-3 seconds to mix the bacteria with the agar. 7. Quickly POUR the mixture onto the pre-warmed plate labeled CONTROL. 8. COVER the plate and lift it from the bench. Holding the plate in your hand, gently rock and rotate the plate in a circular direction. This disperses the COLORTOP agar evenly across the surface of the agar plate. 9

10 EXPLORING THE INFECTIOUS NATURE OF VIRUSES EDVO-Kit 209 Using the Plaque Assay to Determine Viral Titer 100µl Bacterial growth µl 10. Roll 2-3 sec Quickly Pour Circular motion 1:10 Virus Dilution COLORTOP 1:10 Tube 1: REPEAT Steps 9-12 for 1:100 1: min Invert & Incubate 24 hrs. 37 C 9. PREPARE the experimental samples. ADD 100 μl of the 1:10 virus dilution and 100 μl of bacterial growth to a fresh tube of COLORTOP Agar. 10. ROLL agar tube between your palms to mix the agar for 2-3 seconds to mix the bacteria and the virus with the agar. 11. Quickly POUR the mixture onto pre-warmed agar plate labeled 1: COVER the plate and lift it from the bench. Holding the plate in your hand, gently rock and rotate the plate in a circular direction. This disperses the COLORTOP agar evenly across the surface of the agar plate REPEAT steps 9-12 for the 1:100, and 1:1000 virus dilutions. Be sure to work quickly to prevent the COLORTOP agar from solidifying. (Refer to Table 2 for specifics.) 14. ALLOW the soft agar to harden for at least 10 minutes, keeping the plates in an upright position. 15. Once the agar has solidified, STACK the plates on top of one another and TAPE them together. LABEL the plates with your initials or group number. 16. PLACE the plates in an inverted position (agar side on top) in a 37 C incubator overnight (24 hours). 17. VISUALIZE the control and dilution plates. PROCEED to Experimental Results and Analysis. WARNING! Bacteria and Phage must be added, mixed, and poured quickly to prevent hardening of the COLORTOP Agar. 10

11 EDVO-Kit 209 EXPLORING THE INFECTIOUS NATURE OF VIRUSES Experimental Results and Analysis RECORD your observations in Table 3. Make note of size, color, and appearance of plaques. Be sure to count each plaque on a dilution plates if possible. Calculate the viral titer using plates that contain between colonies. Table 3: Observations Plate Observations Number of Plaques Control Plate Plate 1:10 Plate 1:100 Plate 1:1000 Use the appropriate dilution plate to calculate the concentration of viral particles (PFU) using the formula below: 11

12 EXPLORING THE INFECTIOUS NATURE OF VIRUSES EDVO-Kit 209 Study Questions 1. What are viruses? How are they different from plants, animals and microbes? 2. What are bacteriophages? 3. Name and summarize the two reproduction pathways of bacteriophages. 4. What is a plaque assay? Why do we need to quantify viruses? 5. In order to determine the viral titer of a T4 phage solution, serial dilutions of the sample were prepared. 100 μl of each dilution was mixed with 3 ml of COLOR- TOP agar containing E.coli and poured onto an agar base plate. After a 24-hour incubation period at 37 C, 150 plaques were observed on the 1:1000 dilution plate. Using this data, calculate the concentration of the bacteriophage (pfu/ml). 12

13 EDVO-Kit 209 EXPLORING THE INFECTIOUS NATURE OF VIRUSES INSTRUCTOR'S GUIDE Instructor's Guide ADVANCE PREPARATION: What to do: Time Required: When? Prepare LB One hour 2-7 days before use Agar Base Plates Prepare Bacteria Host Culture hours Night before the lab Aliquot PBS 15 minutes Day before the lab DAY OF THE EXPERIMENT: What to do: Time Required: When? Equilibrate waterbath at 60 C and incubator at 37 C Warm LB agar plates in 37 C incubator 10 minutes 60 minutes 1.5 to 2 hours before performing the experiment 1 hour before performing the experiment Preparation of Virus Stock Solution 15 minutes Day of the lab Preparation of COLORTOP Agar 1.5 hours 1.5 hours before lab Inc., all rights reserved

14 INSTRUCTOR'S GUIDE EXPLORING THE INFECTIOUS NATURE OF VIRUSES EDVO-Kit 209 Pre-Lab Preparations - Advance Preparation POUR AGAR BASE PLATES If plates are prepared 2-7 days before use, they must be stored in a sealed plastic bag at 4 C Loosen :60 Agar Agar 60 C 10 ml Large source plates 1. BREAK solid ReadyPour LB Agar into small chunks by vigorously squeezing and shaking the plastic bottle. 2. LOOSEN, but DO NOT REMOVE, the cap on the ReadyPour Agar bottle. This allows the steam to vent during heating. CAUTION: Failure to loosen the cap prior to heating may cause the bottle to break or explode. 3. MICROWAVE the ReadyPour Agar on high for 60 seconds to melt the agar. Carefully REMOVE the bottle from the microwave and MIX by swirling the bottle. Continue to HEAT the solution in 30-second intervals until the agar is completely dissolved (the amber-colored solution should be clear and free of small particles). 4. COOL the ReadyPour Agar to 60 C with careful swirling to promote even dissipation of heat. 5. POUR 10 ml of the cooled ReadyPour Agar into each of the large petri dishes using a 10 ml pipet and pipet pump. 6. COVER and WAIT at least twenty minutes for the LB-agar plates to solidify. For optimal results, leave plates at room temperature overnight. 7. STORE plates at room temperature for no more than two days. Plates should be inverted and placed in a sealable plastic bag to ensure that they do not dry out. Wear Hot Gloves and Goggles during all steps involving heating. NOTE for Step 3: Use extra care and make sure the agar does not boil out of the bottle. Pay close attention and stop the heating if it starts to bubble up. NOTE: If plates are prepared more than two days before use, they should be stored inverted in a plastic bag in the refrigerator (4 C). Remove the plates from the refrigerator and warm in a 37 C incubator for 60 minutes before use. On the day of the experiment, place agar base plates for students in 37 C incubator to warm for at least one hour before performing the experiment. The plates MUST be warm for the experiment to work properly. Quick Reference: Pouring LB Agar Plates Use a sterile 10 ml pipet with a pipet pump to transfer the designated volume of medium to each petri plate. Pipet carefully to avoid forming bubbles. Rock the petri plate back and forth to obtain full coverage. If the molten medium contains bubbles, they can be removed by passing a flame across the surface of the medium. Cover the petri plate and allow the medium to solidify. 14

15 EDVO-Kit 209 EXPLORING THE INFECTIOUS NATURE OF VIRUSES INSTRUCTOR'S GUIDE Pre-Lab Preparations - Advance Preparation Preparation of Bacterial Host Culture 1. Using a sterile loop, ADD two BactoBeads to 10 ml of LB in a 50 ml screw-cap tube. 2. INCUBATE for hours in 37 C incubator. 3. DISPENSE 600 μl of the bacterial culture into ten 1.5 ml snap-top tubes. Pre-Lab Preparations - Day of the Experiment Each Group Should Receive: 4 LB-agar Base Plates, pre-warmed at 37 C 4 15 ml Conical tubes containing 5 ml COLORTOP Agar ml snap-top microcentrifuge tubes ml snap-top microcentrifuge tube containing 1 ml 1X PBS ml snap-top microcentrifuge tube containing 100 μl Bacteriophage Stock Solution ml snap-top microcentrifuge tube containing 600 μl E. coli culture Adjustable volume micropipet COLORTOP Agar Preparation 1. MICROWAVE the COLORTOP Agar on high for 60 seconds to melt the agar. Carefully REMOVE the bottle from the microwave and MIX by swirling the bottle. Continue to HEAT the solution in 30-second intervals until the agar is completely dissolved (the red-colored solution should be clear and free of small particles). Once melted, DISPENSE 5 ml of agar per 15 ml screw-top conical tube (4 tubes per group, 40 tubes total). 2. PLACE tubes of melted agar in 60 C waterbath to keep the agar from solidifying. The agar MUST be kept warm at all times. Bacteriophage Stock Solution Preparation 1. Using a sterile disposable loop, ADD 5 phage beads into 10 ml sterile PBS in a 15 ml conical tube. 2. VORTEX thoroughly. This is the 1X stock solution. 3. ALIQUOT 100 μl for each group. PBS Preparation 1. LABEL ten 1.5 ml snap-top microcentrifuge tubes with 1X PBS. 2. ALIQUOT 1 ml 1X PBS into the microcentrifuge tubes. Each group will receive one. 15

16 INSTRUCTOR'S GUIDE EXPLORING THE INFECTIOUS NATURE OF VIRUSES EDVO-Kit 209 Experiment Results and Analysis Sample Calculation: The 1:100 dilution plate (above) has 50 plaques. 100 μl of the bacteriophage dilution was added to the E. coli culture. Average # of plaques Dilution factor X Volume of diluted virus (in ml) = PFU per ml 50 plaques 0.01 (equals 1:100 dilution) X 0.1 ml of the diluted virus = 50,000 PFU per ml 16

17 Please refer to the kit insert for the Answers to Study Questions

18 APPENDICES EXPLORING THE INFECTIOUS NATURE OF VIRUSES EDVO-Kit 209 Appendices A EDVOTEK Troubleshooting Guide Safety Data Sheets can be found on our website: 18 Inc., all rights reserved

19 EDVO-Kit 209 EXPLORING THE INFECTIOUS NATURE OF VIRUSES APPENDICES Appendix A EDVOTEK Troubleshooting Guides PROBLEM: CAUSE: ANSWER: Bacterial lawn does not form. Plaques do not form. Excessive heat inactivated the virus or killed the bacteria. The COLORTOP agar should be cooled to 60 C before adding the phage/bacteria mixture. Plaques concentrated in one area of the plate. Phage/bacteria solution not thoroughly mixed into the COLORTOP agar before the overlay was poured. COLORTOP agar was not spread evenly across the surface of the plate. Base plate was not properly warmed before pouring the COLORTOP agar. Be sure to spread the COLORTOP agar across the entire surface of the agar base plate by moving the plate in a circular direction. The COLORTOP agar remains liquid for a longer period of time when the base plate is heated, making it easer to spread the agar. Plaques are all different shapes and sizes. Natural Variation Although the majority of plaques should be round, they can take differnent shapes. Count all distinct regions of clearing regardless of the shape and size. Lumpy, bubbly and/or uneven overlay COLORTOP agar was below 60 C when it was poured on the plate. Base plate was not properly warmed before pouring the COLORTOP agar. COLORTOP agar should be warm enough to avoid hardening while adding to the plate to avoid lumps and bubbles in agar. COLORTOP agar may solidify too quickly if it comes into contact with a cold base plate. This makes it difficult to evenly spread the agar. Inc., all rights reserved