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IF YOU CAN SEE THIS MESSAGE YOU ARE NOT IN “SLIDE SHOW” MODE. PERFOMING THE LAB IN THIS MODE WILL NOT ALLOW FOR THE ANIMATIONS AND INTERACTIVITY OF THE EXERCISE TO WORK PROPERLY. TO CHANGE TO “SLIDE SHOW” MODE YOU CAN CLICK ON “VIEW” AT THE TOP OF THE PAGE AND SELECT “SLIDE SHOW” FROM THE PULL DOWN MENU. YOU CAN ALSO JUST HIT THE “F5” KEY. Agar Plates Antiseptic Dispenser Swabs Pencil Microbe Samples Bunsen burner Incubator 37 0C Loops Loops Click on the blackboard to view a larger board for discussion. Click on the INDEX link below to advance directly to a staining lab. Agar Plates pH = 7 pH = 9 BACTERIAL STAINING Most bacteria are difficult to see under the bright field of a microscope. Bacteria are almost colorless (composed primarily of water) and therefore show little contrast with the suspension. To visualize bacteria, either dyes or stains are used. Since staining of bacterial cells is relatively fast, inexpensive, and simple, it is the most commonly used technique to visualize bacterial cells. Staining not only makes bacteria more easily seen, but it allows their morphology (e.g. size and shape) to be visualized more easily. In some cases, specific stains can be used to visualize certain structures (flagella, capsules, endospores, etc) of bacterial cells. pH = 11 Incubator pH = 5 pH = 3 There are several staining methods that are used routinely with bacteria. These methods may be classified as 1) simple and 2) differential. Simple stains will react with all microbes in an identical fashion. They are useful solely for increasing contrast so that morphology, size, and arrangement of organisms can be determined. Differential stains give varying results depending on the organism being treated. These results are often helpful in identifying the microbe. Commonly used microbiological stains generally fall into one of two categories there -10 C 0 -Cbasic stains or acidic 35 C stains (although 50 C 100 C are a few stains such as India Ink which are neutral). Agar Plates pH = 7 pH = 9 pH = 11 Freezer 0 pH = 5 pH = 3 Refrigerator 0 Incubator 0 Incubator 0 Incubator 0 A basic dye is a stain that is positively charged and will therefore react with material that is negatively charged. Bacterial cells have a slight negative charge will therefore attract and bind with basic dyes. Some examples of basic dyes are crystal violet, safranin, basic fuchsin and methylene blue. Acid dyes are negatively charged and are repelled by the bacterial surface forming a deposit around the organism. They stain the background and leave the microbe transparent. Nigrosine and congo red are examples of acid dyes. Agar Plates pH = 7 pH = 9 pH = 11 Freezer -10 0C pH = 5 pH = 3 Refrigerator 0 0C Incubator Incubator Incubator 35 0C 50 0C 100 0C At first glance, the easiest way to stain bacterial cells would appear to be simply mixing the bacterial suspension with the dye and making a wet mount of this mixture. Unfortunately, if you were to try staining bacterial cells in this manner you would find that there was too much background (unbound dye) to allow for visualization of the cells. Therefore, you need to remove the unbound dye. Simply washing off the dye would result in removal of the cells along with the excess dye. Agar Plates pH = 7 pH = 9 pH = 11 Freezer -10 0C pH = 5 pH = 3 Refrigerator 0 0C Incubator Incubator Incubator 35 0C 50 0C 100 0C Therefore, you need a mechanism to fix (adhere) the cells to the slide before staining to allow for removal of excess dye while keeping the cells on the slide. A simple method is that of air drying and heat fixing. The organisms are heat fixed by passing an air-dried smear of the organisms through the flame of a gas burner. The heat coagulates the organisms' proteins causing the bacteria to stick to the slide. Be very careful not to over heat the organisms when fixing them to a slide. This distorts the sample of the organisms. Agar Plates pH = 7 pH = 9 pH = 11 Freezer -10 0C pH = 5 pH = 3 Refrigerator 0 0C Incubator Incubator Incubator 35 0C 50 0C 100 0C Keep in mind the analogy of a fried egg. When you drop a raw egg onto a cold frying pan, it has a certain shape. Start heating it and the proteins (albumin) on the lower surface of the egg precipitate and fix the egg to the pan. At this point there has been a minimal distortion in the shape of the egg as a whole; only a small percent of the proteins have been precipitated. If you keep applying heat, the shape of the entire egg will change and eventually it will be reduced to charred remains. When you heat fix a slide, you want to apply enough heat to precipitate proteins to allow the cells 50 toCstick to 100 theC -10 C 0 the C 35 C slide but not to drastically change the shape of the cells (or reduce them to charred remains). Agar Plates pH = 7 pH = 9 pH = 11 Freezer 0 pH = 5 pH = 3 Refrigerator 0 Incubator 0 Incubator 0 Incubator 0 Observe the links given below to bring you to the VIRTUAL LAB that you wish to perform. If you have performed all of the exercises, you can click on END LAB. Agar Plates pH = 7 pH = 9 pH = 11 pH = 5 pH = 3 Simple Stain Freezer Refrigerator Endospore Stain -10 0C 0 0C Negative Stain Incubator Incubator Gram Stain 35 0C 50 0C Capsule Stain End Lab Acid Fast Stain Incubator 100 0C Agar Plates pH = 7 pH = 9 SIMPLE STAIN Staining is a technique that is used to enhance contrast in a microscopic image. Staining can also be used to highlight structures for viewing. The simple stain can be used to determine cell shape, size, and arrangement. The simple stain is a very simple staining procedure involving only one stain. The most common stains (dyes) used are methylene blue, Gram safranin, and Gram crystal violet. Basic stains, such as methylene blue, Gram0 Csafranin, or Gram crystal violet are useful -10 C 35 C 50 C 100for C staining most bacteria. These stains will readily give up a OH- ion or accept a H+ ion, which leaves the stain positively charged. These positively charged stains adhere readily to the cell surface, since the surface of most bacteria are negatively charged. pH = 11 Freezer 0 pH = 5 pH = 3 Refrigerator 0 Incubator 0 Incubator 0 Incubator 0 SIMPLE STAIN PROCEDURE 1) Perform a bacterial smear of the given bacterial culture 2) Allow the smear to dry thoroughly. 3) Heat-fix the smear cautiously by passing the underside of the slide through the burner flame two or three times. Overheating can distort the cells. -10 C 0 C 35 C 50 C 100 C 4) Saturate the smear with basic dye and let sit for approximately 1 minute. We will use methylene blue 5) Rinse the slide gently with water 6) Observe the slide under the microscope Agar Plates pH = 7 pH = 9 pH = 11 Freezer 0 pH = 5 pH = 3 Refrigerator 0 Incubator 0 Incubator 0 Incubator 0 India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the SLIDES to bring one of them to the rinse station. Now click on the EYE DROPPERS to transfer 1 drop of the bacterial broth to the slide. Click on NEXT when you have allowed the sample to dry on the slide. India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the BUNSEN BURNER to bring the burner to the table. Next click on the slide to fix the bacterial sample to the slide by passing the slide through the flame two or three times. Click on NEXT when the sample has been fixed. India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the bottle of methylene blue dye in the shelves and flood the sample. Let the dye and sample sit for 1 minute. Next click on the water rinse bottle to rinse the dye from the sample. Let air dry. Click on NEXT when the sample has dried. India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the microscope to bring the microscope to the table. Next click on the slide on the rinse table to bring the slide to the microscope stage. Click on NEXT when the slide is on the stage. India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator Loops Water Rinse Bunsen burner Carbolfuchsin You are viewing bacteria from a Simple Stain with methylene blue. Click on the EYEPIECE of the microscope to view the slide under High Power (400 X). You will need to sketch a few of the bacteria cells that you are viewing. Be sure to indicate any colors you observe by using colored markers (pens or pencils) or by indicating any color with a label. 37 0C Click Here go to the next staining procedure Agar Plates pH = 7 pH = 9 ENDOSPORE STAIN The endospore stain is a differential stain used to visualize bacterial endospores. Endospores are formed by some bacteria, such as Bacillus. By forming spores, bacteria can survive in hostile conditions. Spores are resistant to heat, dessication, chemicals, and radiation. Bacteria can form endospores in approximately 6 to 8 hours after being exposed to adverse conditions. The normally growing cell that forms the endospore is called a vegetative cell. Spores are metabolically inactive and -10 C 0 C They can remain viable 35 C 50 C 100 C dehydrated. for thousands of years. When spores are exposed to favorable conditions, they can germinate into a vegetative cell within 90 minutes. pH = 11 Freezer 0 pH = 5 pH = 3 Refrigerator 0 Incubator 0 Incubator 0 Incubator 0 Endospores can form within different areas of the vegetative cell. They can be central, subterminal, or terminal. Central endospores are located within the middle of the vegetative cell. Terminal endospores are located at the end of the vegetative cell. Subterminal endospores are located between the middle and the end of the cell. Endospores can also be larger or smaller in diameter than the vegetative cell. Those that are larger in diameter will produce an area of "swelling" in the vegetative cell. These endospore characteristics are consistent the spore-forming species -10 C 0 within C 35 C 50 C and can 100be C used to identify the organism. Agar Plates pH = 7 pH = 9 pH = 11 Freezer 0 pH = 5 pH = 3 Refrigerator 0 Incubator 0 Incubator 0 Incubator 0 Because of their tough protein coats made of keratin, spores are highly resistant to normal staining procedures. The primary stain in the endospore stain procedure, malachite green, is driven into the cells with heat and readily adheres to the endospore. Since malachite green is water-soluble and does not adhere well to the cell, and since the vegetative cells have been disrupted by heat, the malachite green rinses easily from the vegetative cells, allowing them to readily take up the counterstain. This allows the endospores to be visible with green stain35and cells -10 C the malachite 0 C C the vegetative 50 C 100 Cto be visible with the counterstain. Agar Plates pH = 7 pH = 9 pH = 11 Freezer 0 pH = 5 pH = 3 Refrigerator 0 Incubator 0 Incubator 0 Incubator 0 ENDOSPORE STAIN PROCEDURE 1) Perform a bacterial smear of the organism you are given to stain 2) Saturate the smear with malachite green 3) Heat the slide gently over a Bunsen burner for 5 minutes 4) Rinse the slide gently with water -10 C 0 C 35 C C 100 C 5) Counterstain with safranin for 250minutes 6) Rinse the slide gently with water 7) Observe the slide under the microscope. Endospores will stain green. Vegetative cells will stain red Agar Plates pH = 7 pH = 9 pH = 11 Freezer 0 pH = 5 pH = 3 Refrigerator 0 Incubator 0 Incubator 0 Incubator 0 India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the SLIDES to bring one of them to the rinse station. Now click on the EYE DROPPERS to transfer 1 drop of the bacterial broth to the slide. Click on NEXT when you have allowed the sample to dry on the slide. India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the bottle of malachite green dye in the shelves and flood the sample. The green dye will be visible in the endospores. Let the dye and sample sit for 1 minute. Click on NEXT when the sample has dried. India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Click on the BUNSEN BURNER to bring the burner to the table. Next click on the slide to fix the bacterial sample to the slide by gently warming the sample above the flame for 5 minutes. Click on NEXT when the sample has been fixed. Carbolfuchsin Timer 5 2 3 4 1 min India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the water rinse bottle to rinse the dye from the sample. Let air dry. Click on the counterstain Safranin to stain the sample for two minutes. The red counterstain will be visible in the vegetative bacteria. After the stain has set click on NEXT. India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the water rinse bottle to rinse the counterstain safranin dye from the sample. Let air dry. After the stain has dried click on NEXT. India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the microscope to bring the microscope to the table. Next click on the slide on the rinse table to bring the slide to the microscope stage. Click on NEXT when the slide is on the stage. India Ink Iodine The green stained bacteria are endospores Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator Loops Water Rinse Bunsen burner Carbolfuchsin You are viewing bacteria from an Endospore stain. Click on the Themicroscope red stained to view EYEPIECE of the bacteria are (400 X). You the slide under High Power vegetative will need to sketch a few of the bacteria cells that you are viewing. Be sure to indicate any colors you observe by using colored markers (pens or pencils) or by indicating any color with a label. 37 0C Click Here go to the next staining procedure Agar Plates pH = 7 pH = 9 CAPSULE STAIN The capsule stain employs an acidic stain and a basic stain to detect capsule production. Capsules are formed by organisms such as Klebsiella pneumoniae. Most capsules are composed of polysaccharides, but some are composed of polypeptides. The capsule differs from the slime layer that most bacterial cells produce in that it is a thick, detectable, discrete layer outside the cell wall. Some capsules have well-defined boundaries, and some have fuzzy, trailing edges. Capsules protect bacteria -10 C 0 C 35 C 50 C allow 100 C from the phagocytic action of leukocytes and pathogens to invade the body. If a pathogen loses its ability to form capsules, it can become avirulent. pH = 11 Freezer 0 pH = 5 pH = 3 Refrigerator 0 Incubator 0 Incubator 0 Incubator 0 Bacterial capsules are non-ionic, so neither acidic nor basic stains will adhere to their surfaces. Therefore, the best way to visualize them is to stain the background using an acidic stain and to stain the cell itself using a basic stain. We will use India ink and Gram crystal violet. This leaves the capsule as a clear halo surrounding a purple cell in a field of black. The medium in which the culture is grown as well as the temperature at which it is grown and the age of the culture will affect capsule formation. Older cultures are more likely to exhibit capsule production. When performing a capsule stain -10 C 0 C 35 C 50 C 100 on C your unknown, be sure the culture you take your sample from is at least five days old. Agar Plates pH = 7 pH = 9 pH = 11 Freezer 0 pH = 5 pH = 3 Refrigerator 0 Incubator 0 Incubator 0 Incubator 0 The ability to produce a capsule is an inherited property of the organism, but the capsule is not an absolutely essential cellular component. Capsules help many pathogenic and normal flora bacteria to initially resist phagocytosis by the host's phagocytic cells. In soil and water, capsules help prevent bacteria from being engulfed by protozoans. Capsules also help many bacteria to adhere to surfaces and thus resist flushing. It also enables many bacteria to form biofilms. A biofilm consists layers of bacterial populations adhering to host cells and embedded in a common -10 C 0 C 35 C capsular 50 Cmass. 100 C Agar Plates pH = 7 pH = 9 pH = 11 Freezer 0 pH = 5 pH = 3 Refrigerator 0 Incubator 0 Incubator 0 Incubator 0 CAPSULE STAIN PROCEDURE Agar Plates pH = 7 pH = 9 1) Place a single drop of India ink on a microscope slide 2) Add the organism to be stained and mix into the drop of India ink 3) Spread the mixture on the slide to a thin layer 4) Allow the film to air dry. DO NOT heat or blot dry. Heat will melt the capsule 5) CSaturate violet 1 minute -10 0 C the slide with crystal 35 C 50 for C 100 C 6) Rinse the slide gently with water 7) Allow the slide to air dry 8) Observe the slide under the microscope. The background will be dark. The bacterial cells will be stained purple. The capsule (if present) will appear clear against the dark background pH = 11 Freezer 0 pH = 5 pH = 3 Refrigerator 0 Incubator 0 Incubator 0 Incubator 0 India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the slides to bring one of the slides to the rinse table. Click on the bottle of India ink dye in the shelves and add 1 drop to the slide. Click on NEXT when the dye has been added to the slide. India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the EYE DROPPERS to transfer 1 drop of the bacterial broth to the slide. Allow the thin film to air dry. Click on NEXT when you have allowed the sample to dry on the slide. India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the Crystal Violet to saturate the sample for 1 minute. Click on the water rinse bottle to rinse the dye from the sample. Let air dry. After the stain has dried click on NEXT. India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the microscope to bring the microscope to the table. Next click on the slide on the rinse table to bring the slide to the microscope stage. Click on NEXT when the slide is on the stage. India Ink Iodine The purple stain is the bacteria Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Loops Water Rinse Bunsen burner Carbolfuchsin You are viewing bacteria from a Capsule stain. Click on the EYEPIECE of the microscope to view The the cleared slide under High areaswill in the Power (1000 X). You need to sketch India ink are the a few of the bacteria cells that you are capsules viewing. Be sure to indicate any colors you observe by using colored markers (pens or pencils) or by indicating any color with a label. Incubator 37 0C Click Here go to the next staining procedure Agar Plates pH = 7 pH = 9 ACID FAST STAIN The acid-fast stain is a differential stain used to identify acid-fast organisms such as members of the genus Mycobacterium. Acid-fast organisms are characterized by wax-like, nearly impermeable cell walls; they contain mycolic acid and large amounts of fatty acids, waxes, and complex lipids. Acid-fast organisms are highly resistant to disinfectants and dry conditions. pH = 11 Freezer -10 0C pH = 5 pH = 3 Refrigerator 0 0C Incubator Incubator Incubator 35 0C 50 0C 100 0C Because the cell wall is so resistant to most compounds, acid-fast organisms require a special staining technique. The primary stain used in acid-fast staining, carbolfuchsin, is lipid-soluble and contains phenol, which helps the stain penetrate the cell wall. This is further assisted by the addition of heat. The smear is then rinsed with a very strong decolorizer, which strips the stain from all non-acid-fast cells but does not permeate the cell wall of acid-fast organisms. The decolorized non-acid-fast cells then take up the counterstain. -10 C 0 C 35 C 50 C 100 C Agar Plates pH = 7 pH = 9 pH = 11 Freezer 0 pH = 5 pH = 3 Refrigerator 0 Incubator 0 Incubator 0 Incubator 0 The acid-fast stain is an especially important test for the genus Mycobacterium. There are two distinct pathogens in this group: M. tuberculosis, the causative organism of tuberculosis, and M. leprae, the causative agent of leprosy. Agar Plates pH = 7 pH = 9 pH = 11 Freezer -10 0C pH = 5 pH = 3 Refrigerator 0 0C Incubator Incubator Incubator 35 0C 50 0C 100 0C ACID FAST STAIN PROCEDURE Agar Plates pH = 7 pH = 9 1) Perform a bacterial smear 2) Flood the smear with carbolfuchsin 3) Heat the slide gently over the Bunsen burner for 5 minutes 4) Rinse the slide gently with water 5) Decolorize the slide with acid-alcohol until the rinse runs clear 6) CRinse the water 50 C -10 0 C slide gently with 35 C 100 C 7) Counterstain with methylene blue for 2 minutes 8) Rinse the slide gently with water 9) Observe the slide under the microscope. Acidfast cells will stain fuchsia (pink or red). Non-acidfast cells will stain blue pH = 11 Freezer 0 pH = 5 pH = 3 Refrigerator 0 Incubator 0 Incubator 0 Incubator 0 India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the SLIDES to bring one of them to the rinse station. Now click on the EYE DROPPERS to transfer 1 drop of the bacterial broth to the slide. Click on NEXT when you have allowed the sample to dry on the slide. India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the bottle of Carbolfuchsin dye in the shelves and flood the smear. Click on NEXT when the smear has been stained. India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Click on the BUNSEN BURNER to bring the burner to the table. Next click on the slide to fix the bacterial sample to the slide by gently warming the sample above the flame for 5 minutes. Click on NEXT when the sample has been fixed. Carbolfuchsin Timer 5 2 3 4 1 min India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the water rinse bottle to rinse the dye from the sample. Let air dry. Next click on the acid-alcohol decolorizer to rinse the stain from the slide. Next click on the rinse bottle again to clear the acid-alcohol. Click on NEXT when finished rinsing. India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the Methylene blue dye in the and saturate the sample for 2 minutes. Next click on the water rinse bottle to rinse the dye from the sample. Let air dry. Click on NEXT when the sample has dried. India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the microscope to bring the microscope to the table. Next click on the slide on the rinse table to bring the slide to the microscope stage. Click on NEXT when the slide is on the stage. India Ink Iodine Acid Fast bacteria will stain red Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator Loops Water Rinse Bunsen burner Carbolfuchsin You are viewing bacteria from an Acid Fast stain. Click on the EYEPIECE of the microscope toNon-acid view thefast slide under High Power (400 X). bacteria You willwill need to sketch a staincells blue that you are few of the bacteria viewing. Be sure to indicate any colors you observe by using colored markers (pens or pencils) or by indicating any color with a label. 37 0C Click Here go to the next staining procedure Agar Plates pH = 7 pH = 9 NEGATIVE STAIN The negative stain is particularly useful for determining cell size and arrangement. It can also be used to stain cells that are too delicate to be heat-fixed. We will use nigrosin as our negative stain. Nigrosin is an acidic stain. This means that the stain readily gives up a H+ ion and becomes negatively charged. Since the surface of most bacterial cells is negatively charged, the cell surface repels the stain. The glass of the slide will stain, but the bacterial cells will not. The bacteria will show up -10 0 C 35 C 50 C 100 C asCclear spots against a dark background. pH = 11 Freezer 0 pH = 5 pH = 3 Refrigerator 0 Incubator 0 Incubator 0 Incubator 0 Negative staining is an excellent way to determine an organism’s cellular morphology. Since the cells themselves are not stained, their morphology is not distorted in any way. The nigrosin provides a dark background against which the shapes of the unstained cells are clearly visible. This method provides a high degree of contrast not available in most other staining procedures. Agar Plates pH = 7 pH = 9 pH = 11 Freezer -10 0C pH = 5 pH = 3 Refrigerator 0 0C Incubator Incubator Incubator 35 0C 50 0C 100 0C NEGATIVE STAIN PROCEDURE Agar Plates 1) Place a single drop of nigrosin on a pH = 7 pH = 9 pH = 11 pH = 5 pH = 3 microscope slide 2) Mix a sample of your organism to stain into the drop of nigrosin 3) Spread the sample on the slide into a thin Freezer layer -10 0C Refrigerator 0 0C Incubator Incubator Incubator 35 0C 50 0C 100 0C 4) Allow the film to air dry 5) Observe the slide under the microscope. The bacteria will be clear and the background will stain dark India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the slides to bring one of the slides to the rinse table. Click on the bottle of Nigrosin dye in the and add 1 drop to the slide. Click on NEXT when the dye has been added to the slide. India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the EYE DROPPERS to transfer 1 drop of the bacterial broth to the slide. Allow the thin film to air dry. Click on NEXT when you have allowed the sample to dry on the slide. India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the microscope to bring the microscope to the table. Next click on the slide on the rinse table to bring the slide to the microscope stage. Click on NEXT when the slide is on the stage. India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator Loops Water Rinse Bunsen burner Carbolfuchsin You are viewing bacteria from a Negative stain. Click on the EYEPIECE of the microscope to view the slide under High Power (1000 X). You will need to sketch a few of the bacteria cells that you are viewing. Be sure to indicate any colors you observe by using colored markers (pens or pencils) or by indicating any color with a label. 37 0C Click Here go to the next staining procedure Agar Plates pH = 7 pH = 9 GRAM STAIN The Gram stain is the most important staining procedure in microbiology. It is used to differentiate between gram positive organisms and gram negative organisms. Hence, it is a differential stain. Gram negative and gram positive organisms are distinguished from each other by differences in their cell walls. These differences affect many aspects of the cell, including the way the cell takes up and retains stains. pH = 11 Freezer -10 0C pH = 5 pH = 3 Refrigerator 0 0C Incubator Incubator Incubator 35 0C 50 0C 100 0C Gram positive cells take up crystal violet, which is then fixed in the cell with iodine mordant. This forms a crystal-violet iodine complex which remains in the cell even after decolorizing. It is thought that this happens because the cell walls of gram positive organisms include a thick layer of protein-sugar complexes called peptidoglycans. This layer makes up 60-90% of the gram positive cell wall. Decolorizing the cell causes this thick cell wall to dehydrate and shrink, which closes the pores in the cell wall and prevents the stain from exiting the cell. of the gram staining procedure, gram -10 C At the0 end C 35 C 50 C 100 C positive cells will be stained a purplish-blue color. Agar Plates pH = 7 pH = 9 pH = 11 Freezer 0 pH = 5 pH = 3 Refrigerator 0 Incubator 0 Incubator 0 Incubator 0 Gram negative cells also take up crystal violet, and the iodine forms a crystal violet-iodine complex in the cells as it did in the gram positive cells. However, the cell walls of gram negative organisms do not retain this complex when decolorized. Peptidoglycans are present in the cell walls of gram negative organisms, but they only comprise 10-20% of the cell wall. Gram negative cells also have an outer layer which gram positive organisms do not have; this layer is made up of lipids, polysaccharides, and proteins. Exposing gram negative cells to the0 Cdecolorizer dissolves -10 C 35 C the lipids 50 Cin the cell 100 C walls, which allows the crystal violet-iodine complex to leach out of the cells. This allows the cells to subsequently be stained with safranin. At the end of the gram staining procedure, gram negative cells will be stained a reddish-pink color. Agar Plates pH = 7 pH = 9 pH = 11 Freezer 0 pH = 5 pH = 3 Refrigerator 0 Incubator 0 Incubator 0 Incubator 0 Often, detecting the presence of microorganisms and determining whether an infection is caused by an organism that is Gram-positive or Gram-negative will be sufficient to allow a doctor to prescribe treatment with an appropriate antibiotic while waiting for more specific tests, such as a culture, to be completed. Agar Plates pH = 7 pH = 9 pH = 11 Freezer -10 0C pH = 5 pH = 3 Refrigerator 0 0C Incubator Incubator Incubator 35 0C 50 0C 100 0C GRAM STAIN PROCEDURE Agar Plates pH = 7 pH = 9 1) Perform a bacterial smear of your given culture 2) Saturate the smear with crystal violet for 1 minute 3) Rinse the slide gently with water 4) Saturate the smear with iodine for 1 minute 5) Rinse the slide gently with water 6) Decolorize with Gram decolorizer for 3-5 seconds 7) CRinse the -10 0 C slide gently with 35 Cwater 50 C 100 C 8) Counterstain with safranin for 1 minute 9) Rinse the slide gently with water 10) Observe the slide under the microscope. Gram positive bacteria will stain purple. Gram negative bacteria will stain red/pink pH = 11 Freezer 0 pH = 5 pH = 3 Refrigerator 0 Incubator 0 Incubator 0 Incubator 0 India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the SLIDES to bring one of them to the rinse station. Now click on the EYE DROPPERS to transfer 1 drop of the bacterial broth to the slide. Click on NEXT when you have allowed the sample to dry on the slide. India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the Crystal Violet to saturate the sample for 1 minute. Click on the water rinse bottle to rinse the dye from the sample. Click on NEXT when you are finished with the rinse. India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the Iodine bottle to saturate the sample for 1 minute. Click on the water rinse bottle to rinse the dye from the sample. Click on NEXT when you are finished with the rinse. India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the Gram Decolorizer bottle to rinse the sample for 2-3 seconds. Next Click on the water rinse bottle to rinse the decolorizer from the sample. Click on NEXT when you are finished with the rinse. India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the counterstain Safranin to stain the sample for 1 minute. Next click on the water rinse bottle to rinse the dye from the sample. Let air dry. After the slide has dried click on NEXT. India Ink Iodine Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Incubator 37 0C Loops Water Rinse Bunsen burner Carbolfuchsin Click on the microscope to bring the microscope to the table. Next click on the slide on the rinse table to bring the slide to the microscope stage. Click on NEXT when the slide is on the stage. India Ink Iodine Gram Positive Bacteria will stain Purple Crystal Violet Methylene Blue Malachite Green Eye Droppers Agar Plates Microscope Safranin Nigrosin Slides Acid Alcohol Gram Decolorizer Loops Water Rinse Bunsen burner Carbolfuchsin You are viewing bacteria from a Gram stain. Click on the EYEPIECE of the Gram the Negative microscope to view slide under High Bacteria will to sketch a Power (400 X). You will need Pink few of the bacteriastain cells that you are viewing. Be sure to indicate any colors you observe by using colored markers (pens or pencils) or by indicating any color with a label. Incubator 37 0C Click Here go to the next staining procedure END