Harini VenkatachalapathiAnna ChenNivedha GanesanElisa Bu ShaHonors Biology Pd 5Heavers/Costello12/11/17IDENTIFICATION OF BACTERIAIntroduction: Microbiology is the scientific study of microorganisms such as bacteria,Protists, viruses, and Fungi. The development of proper microbiological techniques is vital to any scientist pursuing a career in microbiology. The microbiologist must be able to isolate and maintain microorganisms under sterile conditions, as well as differentiate and identify specific microorganisms. Proper handling, thorough knowledge of staining techniques and microscopy skills is of utmost importance to any scientist working in a microbiology laboratory.Purpose: To understand and use proper microbiological methods. To identify bacteriausing Methylene Blue and Gram staining.List:Serratia marcescens Gram negative rodBacillus cereus Gram positive, rodRhodospirillum rubrum Gram negative, spiralBranhamella catarrhalis Gram negative, coccusMicrococcus luteus Gram positive coccusMaterials: For each Lab group2 Petri dishes with unknown bacteria labeled A-EInoculating loop 2Microscope slides 4Clothespins 2-4Methylene Blue stainSafranin O stain for Gram stainCrystal violetGram’s Iodine solution (Potassium iodide)Colored pencils95% ethyl alcoholPermanent markersDistilled water and droppersAprons and goggles250 ml beakerSpray bottle containing 10% bleachSafety Precautions: Wear closed-toed shoes. Tie back loose hair; wear an apron and goggles before you begin.Procedure: Day 1To make sure that you are working in sterile conditions, spray some bleach solution on the lab table and wipe it up with a clean wad of paper towels, wash your hands and begin.Slide making technique:Each Lab group will work with two of the above unknowns. They will then share their results with the other groups.Obtain 2 Petri dishes from your teacher labeled A –E.Without opening the plates, turn them upside down and draw the pattern of growth you see for each of the unknowns in your data table. Label the bacteria lawn and a bacteria colony.With the permanent marker label two clean microscope slides as A1 and A2 (if the unknown is A) and two slides as B1 and B2 (if the unknown is B) etc. Include your group # and period # on each slide.Prepare a bacterial smear on the slide labeled A as follows:Place a drop of distilled water in the center of one of the slides.Light the Bunsen burner (make sure that you use the safety precautions that your teacher has demonstrated)Flame the inoculating loop (the loop should be red hot) and allow to cool for 10 sec (Count “one thousand one, one thousand two… till 10)Remove ONE isolated colony from unknown plate A by gently dragging the loop over the colony on the plate (swoop not scoop!)Close the plates when done.Rub the inoculating loop containing the bacteria across the drop of water on the microscope slide.Spread the water into a thin film across the slide. Do not smear your label.Place the slide on a clean paper towel in the middle of the table and allow to air dry.Sterilize the inoculating loop by flaming it as in c aboveAs the first slide is drying continue the above procedure (a to i) for the A2 slide.Repeat the procedure for the B slides.Two students can work on slide A and the other two on slide B.You should turn off the Bunsen burner if the slides are not dried at this time. Turn it back on when all four slides have dried and you can do #6 with all the slides at the same time.After the slides have dried, place a clothespin on one end of each slide and fix the bacteria to the slide by passing it back and forth through an open flame several times (a maximum of 6 times back and forth should be sufficient)NOTE: DO NOT HOLD THE SLIDE WITH YOUR HANDS WHILE DOING THIS OR YOU MAY GET BURNED. DO NOT HOLD THE SLIDE OVER THE OPEN FLAME: BE SURE TO PASS IT THROUGH.Each group should have two slides for each unknown they have been given.Place your slides on the tray. Make sure that the label is still intact.Clean up and return other equipment to the cart. Spray the table with bleach; wipe dry and wash hands with soap before you leave the class.Day 2Simple Staining with Methylene BluePlace a clothespin on one end of a slide labeled A1. Hold it over the sink.Place several drops of Methylene Blue on the slide so that it covers the entire slide.Allow the stain to react for 45 seconds.Tilt the slide to allow the excess stain to drain. Rinse the slide by dipping it in the 250ml beaker of water several times.BLOT the slide dry with paper towels (ask your teacher to demonstrate). Do not wipe dry or you will wipe the bacterial smear off the slide.Repeat with slide B1.Gram Stain MethodPlace the clothespin on the end of the slide labeled A2 and hold it over the sink.Place several drops of crystal violet stain on the slide so as to cover the entire surface of the slide.Allow the stain to react for 60 sec.Tilt the slide to drain the stain. Rinse by dipping it into a beaker of clean water.Hold the slide again over the sink and add several drops of Gram stain.Allow staining for 60 sec. Rinse the slide again in a clean beaker of water.Decolorize the smear with 95% alcohol. Tilt the slide and apply the alcohol drop by drop above the smear allowing it to run across the surface of the slide till the alcohol runs clear.Rinse the slide in a beaker of clear water.Hold the slide level again over the sink and apply several drops of Safranin (1%)Allow staining for 30 sec.Drain and rinse the slide with water as in step 4 above.BLOT the slide dry with paper towelsRepeat for each of the other #2 slides.Clean up/Observation:Place all materials and stains in the tub. Return the tub to the cart.Wipe down your lab table with a damp paper towel (in case you have spilled stains).Take out the microscopes. Examine the slides under the microscope. Draw and color a few cells.Slides are Gram-positive if they appear purple, Gram-negative cells are pink.Once you are done with your slides, exchange them with the other 3 unknowns from other groups and draw those cells too. You should have data for a total of 5 slides. Turn in your slides to your teacher for proper disposal.Data Table:Analysis:Did the simple methylene blue staining techniques allow you to differentiate between the different bacteria? ExplainThe methylene blue staining technique didn’t really help in differentiating between the different bacteria but it did help with defining the shape of the bacteria. It stained the edges and parts of the bacteria making the shape of the bacteria more visible when seen under the microscope.Did the gram staining technique allow you to differentiate between the cells? ExplainThe gram staining method allowed us to differentiate between the cells by staining the bacteria a particular color like either pink or purple depending on what bacteria it was and whether it was positive or negative. This allowed us to identify the shape and the name of the bacteria.Identify each of your unknowns using the ID information given. Place your answer on the data table.A: Serratia Marcescens Gram Negative RodB: Branhamella Catarrhalis Gram Negative CoccusC: Rhodospirillum Rubrum Gram Negative SpiralD: Bacillus Cereus Gram Positive RodE: Micrococcus Luteus Gram Positive CoccusOur purpose for this exercise understand and use proper microbiological methods and to identify bacteria using methylene blue and gram staining. I feel like my group and I fulfilled this purpose as we successfully stained the bacteria without many difficulties except for the part were the bottle containing safranin had a clogged nozzle and kind of exploded on mostly me when we tried to unclog it. I think we also used proper microbiological methods as we followed the instructions as said and because we had it memorized we didn’t have unnecessary breaks just to read the instructions and we also had clear workspace not too messy.What we did:I started the bunsen burner, at the start it went too high and I kind of got confused in how to bring it down but then we figured it out and got the flame at the right height. Then Elisa went out to get the tray with the things and then Anna got the bacteria culture. We then set up everything to make sure we were ready and then Anna and Elisa worked on the slide for C1 and C2 while Nivedha and I worked on the slide for D1 and D2. While smearing the bacteria with water on the slide it was hard to get a smooth texture, but it was great that all of us had the procedure memorized and the thing did run smoothly. After all of us were done smearing the bacteria on the slides and had let them dry, we started to pass them over the flame, Elisa and Nivedha were great at passing the slide over the flames as they got both the water evaporated and did not have the marshmallow effect, while on the other hand Anna and me got the marshmallow effect on the slide because we could have held it in the flame too long. In the end, though all was good.Conclusion:What characteristics of bacteria did you observe in this exercise?In this exercise, we observed the shape of certain bacteria and its color depending on the way it reacted to the stains. The five bacteria that our class used appeared either purple or pink after the staining depending on what bacteria it was. This allowed us to determine whether the bacteria were gram-positive or if it was gram negative.Research each of the bacteria that you identified and describe their place in the ecosystem.The bacteria that my group and I identified were Rhodospirillum Rubrum and Bacillus Cereus. All the bacteria that we identified as a group is Serratia marcescens, Bacillus cereus, Rhodospirillum rubrum, Branhamella catarrhalis and Micrococcus luteus.Rhodospirillum rubrum: Gram-Negative Spiral from the family Rhodospirillaceae.Bacillus cereus: Is the type of bacteria that produce toxins. It is a gram-positive rod commonly found in soil and foods or other prepared foods that have sat out too long at room temperature, it comes from the family of Bacillaceae.Serratia Marcescens: A gram-negative rod from the family Enterobacteriaceae. The most dominant mode of Serratia marcescens spread is thought to be hand-to-hand transmission by hospital personnel.(23, 68). Antimicrobe.org.Micrococcus Luteus: It is a gram-positive coccus from the family Micrococcaceae and it is urease and catalase positive. This micrococcus luteus can grow well in environments just with little water or high salt concentrations.Branhamella catarrhalis: A gram-negative cocci from the family Branhamaceae. It causes infections of the respiratory system, middle ear, eye, central nervous system, and joints of humans.Do they cause diseases? ExplainYes they do, for example Rhodospirillum Rubrum causes diseases like Tinea (ringworm) and Onychomycosis (nail fungus), Bacillus Cereus causes diarrhea, nausea, and vomiting, Micrococcus luteus causes septic shock which is not recorded in medical literature, Branhamella Catarrhalis causes bronchitis and pneumonia in children, chronic lung disease in adults. Serratia Marcescens cause diseases like respiratory tract infections and pneumonia.Our sources of error include having the marshmallow effect on the slide after passing it over the flame, an unpredictable fire not knowing whether it will move or if it is going to get higher, and we could have also contaminated the bacteria with our breath without even knowing we were doing so. Our next step includes being more careful while we are close to the bacteria, like holding our breaths while opening the Petri dishes containing the bacteria as not to contaminate anything. We also have to be careful while staining the bacteria as we could overstain it.