Steak plate method for isolation of bacteria—modified for home preparation during COVID READ THIS WHOLE DOCUMENT FIRST, BEFORE YOU BEGIN ANY WORK! GOAL: the intention is to allow student to gain some experience and familiarity with inoculating a petri plate in order to isolate the species from a body sample. Students will use tryptic soy agar plates (college-provided) and cotton swabs (student-provided) to practice inoculating (=placing the sample on the plate) body samples collected using a cotton swab. ABOUT THE PETRI PLATE: Petri dishes are special plastic containers that in Microbiology will be made into Petri plates when sterilized agar media are poured in and allowed to solidify. They consist of a top and bottom, the bottom being where the agar medium is poured, and the top is the lid. The lid is not tight fitting to allow for easy opening and closing during the inoculation process. The lid simply sits by gravity over the plate bottom (containing the agar) to prevent things from falling on the agar surface. If that thing is a microbe, the lid protects against growth of that unwanted introduced microbe; microbes we do not intentionally put on media for inoculation are called contaminants. You could think of contaminants like weeds in your garden--they just seem to pop up, and were not put there, and are, therefore, unwanted! Plates are always stored/placed upside down—this prevents any condensation (that forms from trapped water vapor) from falling on the agar surface. Wet agar surfaces don’t allow bacteria to grow at all, or to grow well (not in the pattern intended). MATERIALS NEEDED: Petri plates, for labeling either permanent marker or tape (masking or painter’s) and pen, rubber bands or tape for securing the plate once inoculated, disposable plastic bag or plastic container (such as boxes that prepared salad mixes can come in), clean cotton swabs, container of boiling water Appropriate body samples to take (only choose these—you will have 4 plates to use so you could do two of the same samples so you have a duplicate set, or choose 4 different samples): front top tooth (incisor) outer surface, between toes, belly button, behind ear, outer nose crease, armpit (DO NOT SAMPLE DEEP INSIDE YOUR BODY like throat or inside your nose, and DO NOT SAMPLE PRIVATE BODY REGIONS!) Steps for inoculating your plates: 1. First, I suggest you use one plate to explore with before you actual take the real samples to inoculate the remaining plates. With this plate, just check it out—figure which side is the top vs. bottom, how the lid works, touch the agar surface to see the texture and firmness of it (it’s ok to do this on this one plate only, it’s your “throw away” plate. Remember that it is sterile so no need to worry about touching the inside surface! Maybe try writing on the plate (outside plastic, see below) with your permanent marker to see how that works. Once you are done exploring, wrap it up tightly and securely in a bag and dispose of it in the trash—do not ever go back to open this plate up. 2. Now, prepare to begin by gathering all of your materials for inoculating your first plate (you will inoculate them one at a time)—you need one plate, your marker or tape and pen (for label), your cotton swab container (do not remove your swab until right before you use it—WHY am I telling you this?), and your container of boiling water (it doesn’t have to be a lot of water because you are only using it to moisten your swab in order to get a good sample, but please be careful because it’s boiling water). 3. Label your plate on the bottom and near the edge (write directly on the plate with your permanent marker, or if using tape, cut a thin, long strip about 1 1/2in long X 1/4in wide). List two informational items on your label, the sample source (body part chosen) and date taken. 4. Now you are ready to gather the sample and inoculate the plate: turn the plate right side up get swab and dip into container of boiling water (do not touch the sides or bottom of container)—you just want to moisten the swab so barely dip it in and when you pull it out, hold it a couple inches from the water surface to let it cool a bit (15 sec) take the body sample by zig-zagging back and forth on the area to transfer the sample on to the swab (you may spin the swab periodically to get a better sample) Immediately inoculate the plate as follows— • tilt the plate lid up in order to touch the swab to the agar surface [“pop” the lid up at an angle only (not completely vertical), but do NOT remove the whole lid or open it all the way or you will likely get contamination] • swab back and forth (zig-zag) on the top 1/3 of the plates (agar) surface—you should overlap your zig- zags and periodically spin the swab to ensure good transfer of collected sample • on the remaining 2/3 of the plate do the following—using the same swab, pull down from the area you just inoculated straight down into the remaining 2/3 of the plate so that you get several lines you pull down perpendicular to your initial zig-zag inoculation (see drawing) • place the lid down over the plate fully, dispose of your swab in the trash, and secure your plate so it cannot open accidentally by either binding it around the middle with a rubber band, or using some tape on the edges—this plate should never be opened, accidentally or intentionally • do the same process with your other plates and different body samples • when all done, incubate your plates in a bag and in a place where it is not too cold or hot. check for growth after 2 days and keep checking everyday after that up to one week and one day. When your growth (roundish dots called colonies) are at least about the size of a sesame seed, you are ready to complete the assigned questions. After you decide they are ready to evaluate, do the following below and then discard as directed below. Do not keep your plates past their use and do not put them in the fridge after inoculation. This is a concentrated sample. Do not allow your pets or children near these! Read on, for what to turn in for your assignment. OBSERVATIONS: How long did growth take to appear? Is the growth typical of bacteria (smooth and paste-like) or mold (hairy and filamentous), or do you see both? if you grew mold, try to ignore it since it is not from your body! Focusing on the bacterial colonies only, based on differences you see in relation to colony size, color, shape, surface attributes (like smooth, shiny, wrinkles, for example), determine the number of different species you have and record here. Do you have good isolation of the different species in your sample (colonies of the different types that are not touching)? You may have some species that are well-isolated while other are not, please note any differences you see. Using the lab manual to identify the correct terminology for colony description, describe one wellisolated colony (or, if you didn’t get good isolation then note the color and surface features only). Write NA if you did not get isolated colonies. IF you didn’t get growth on some or all of your plates, explain what you think are the possible reasons why you didn’t get growth. Tell us about your overall experience— Were you successful? (how do you know?) Were there any things you would do differently? Did you make mistakes? Did your agar crack? Did you make improvements after the first plate you inoculated--what adjustments did you make? Did you follow the steps in the right order, or did you forget anything? Please share any final thoughts about this experience of streak plating. You must include, along with this completed document, pictures of your labeled plates (your labels must be clear and visible in the pictures) BEFORE incubation (growing them). You must include pictures of those same plates (your labels must be clear and visible in the pictures) AFTER incubation (growing them). Make sure that the growth is visible in the pictures if growth occurs. Include pictures of ALL plates, even the ones that did not grow any microbes. PROPER PLATE DISPOSAL—once you make your observations and take your photographs, wrap up your plates so they cannot be opened, put in your ziplock bag and place in another bag and tape that bag shut. Place it in a secure outdoor trash bin so that no person or animal can get into them! Figures from https://greenbioresearch.com/culture-bacteria-using-pre-made-nutrient-agar-plates/ Steak plate method for isolation of bacteria—modified for home preparation during COVID READ THIS WHOLE DOCUMENT FIRST, BEFORE YOU BEGIN ANY WORK! GOAL: the intention is to allow student to gain some experience and familiarity with inoculating a petri plate in order to isolate the species from a body sample. Students will use tryptic soy agar plates (college-provided) and cotton swabs (student-provided) to practice inoculating (=placing the sample on the plate) body samples collected using a cotton swab. ABOUT THE PETRI PLATE: Petri dishes are special plastic containers that in Microbiology will be made into Petri plates when sterilized agar media are poured in and allowed to solidify. They consist of a top and bottom, the bottom being where the agar medium is poured, and the top is the lid. The lid is not tight fitting to allow for easy opening and closing during the inoculation process. The lid simply sits by gravity over the plate bottom (containing the agar) to prevent things from falling on the agar surface. If that thing is a microbe, the lid protects against growth of that unwanted introduced microbe; microbes we do not intentionally put on media for inoculation are called contaminants. You could think of contaminants like weeds in your garden--they just seem to pop up, and were not put there, and are, therefore, unwanted! Plates are always stored/placed upside down—this prevents any condensation (that forms from trapped water vapor) from falling on the agar surface. Wet agar surfaces don’t allow bacteria to grow at all, or to grow well (not in the pattern intended). MATERIALS NEEDED: Petri plates, for labeling either permanent marker or tape (masking or painter’s) and pen, rubber bands or tape for securing the plate once inoculated, disposable plastic bag or plastic container (such as boxes that prepared salad mixes can come in), clean cotton swabs, container of boiling water Appropriate body samples to take (only choose these—you will have 4 plates to use so you could do two of the same samples so you have a duplicate set, or choose 4 different samples): front top tooth (incisor) outer surface, between toes, belly button, behind ear, outer nose crease, armpit (DO NOT SAMPLE DEEP INSIDE YOUR BODY like throat or inside your nose, and DO NOT SAMPLE PRIVATE BODY REGIONS!) Steps for inoculating your plates: 1. First, I suggest you use one plate to explore with before you actual take the real samples to inoculate the remaining plates. With this plate, just check it out—figure which side is the top vs. bottom, how the lid works, touch the agar surface to see the texture and firmness of it (it’s ok to do this on this one plate only, it’s your “throw away” plate. Remember that it is sterile so no need to worry about touching the inside surface! Maybe try writing on the plate (outside plastic, see below) with your permanent marker to see how that works. Once you are done exploring, wrap it up tightly and securely in a bag and dispose of it in the trash—do not ever go back to open this plate up. 2. Now, prepare to begin by gathering all of your materials for inoculating your first plate (you will inoculate them one at a time)—you need one plate, your marker or tape and pen (for label), your cotton swab container (do not remove your swab until right before you use it—WHY am I telling you this?), and your container of boiling water (it doesn’t have to be a lot of water because you are only using it to moisten your swab in order to get a good sample, but please be careful because it’s boiling water). 3. Label your plate on the bottom and near the edge (write directly on the plate with your permanent marker, or if using tape, cut a thin, long strip about 1 1/2in long X 1/4in wide). List two informational items on your label, the sample source (body part chosen) and date taken. 4. Now you are ready to gather the sample and inoculate the plate: turn the plate right side up get swab and dip into container of boiling water (do not touch the sides or bottom of container)—you just want to moisten the swab so barely dip it in and when you pull it out, hold it a couple inches from the water surface to let it cool a bit (15 sec) take the body sample by zig-zagging back and forth on the area to transfer the sample on to the swab (you may spin the swab periodically to get a better sample) Immediately inoculate the plate as follows— • tilt the plate lid up in order to touch the swab to the agar surface [“pop” the lid up at an angle only (not completely vertical), but do NOT remove the whole lid or open it all the way or you will likely get contamination] • swab back and forth (zig-zag) on the top 1/3 of the plates (agar) surface—you should overlap your zig- zags and periodically spin the swab to ensure good transfer of collected sample • on the remaining 2/3 of the plate do the following—using the same swab, pull down from the area you just inoculated straight down into the remaining 2/3 of the plate so that you get several lines you pull down perpendicular to your initial zig-zag inoculation (see drawing) • place the lid down over the plate fully, dispose of your swab in the trash, and secure your plate so it cannot open accidentally by either binding it around the middle with a rubber band, or using some tape on the edges—this plate should never be opened, accidentally or intentionally • do the same process with your other plates and different body samples • when all done, incubate your plates in a bag and in a place where it is not too cold or hot. check for growth after 2 days and keep checking everyday after that up to one week and one day. When your growth (roundish dots called colonies) are at least about the size of a sesame seed, you are ready to complete the assigned questions. After you decide they are ready to evaluate, do the following below and then discard as directed below. Do not keep your plates past their use and do not put them in the fridge after inoculation. This is a concentrated sample. Do not allow your pets or children near these! Read on, for what to turn in for your assignment. OBSERVATIONS: How long did growth take to appear? Is the growth typical of bacteria (smooth and paste-like) or mold (hairy and filamentous), or do you see both? if you grew mold, try to ignore it since it is not from your body! Focusing on the bacterial colonies only, based on differences you see in relation to colony size, color, shape, surface attributes (like smooth, shiny, wrinkles, for example), determine the number of different species you have and record here. Do you have good isolation of the different species in your sample (colonies of the different types that are not touching)? You may have some species that are well-isolated while other are not, please note any differences you see. Using the lab manual to identify the correct terminology for colony description, describe one wellisolated colony (or, if you didn’t get good isolation then note the color and surface features only). Write NA if you did not get isolated colonies. IF you didn’t get growth on some or all of your plates, explain what you think are the possible reasons why you didn’t get growth. Tell us about your overall experience— Were you successful? (how do you know?) Were there any things you would do differently? Did you make mistakes? Did your agar crack? Did you make improvements after the first plate you inoculated--what adjustments did you make? Did you follow the steps in the right order, or did you forget anything? Please share any final thoughts about this experience of streak plating. You must include, along with this completed document, pictures of your labeled plates (your labels must be clear and visible in the pictures) BEFORE incubation (growing them). You must include pictures of those same plates (your labels must be clear and visible in the pictures) AFTER incubation (growing them). Make sure that the growth is visible in the pictures if growth occurs. Include pictures of ALL plates, even the ones that did not grow any microbes. PROPER PLATE DISPOSAL—once you make your observations and take your photographs, wrap up your plates so they cannot be opened, put in your ziplock bag and place in another bag and tape that bag shut. Place it in a secure outdoor trash bin so that no person or animal can get into them! Figures from https://greenbioresearch.com/culture-bacteria-using-pre-made-nutrient-agar-plates/ Steak plate method for isolation of bacteria—modified for home preparation during COVID READ THIS WHOLE DOCUMENT FIRST, BEFORE YOU BEGIN ANY WORK! GOAL: the intention is to allow student to gain some experience and familiarity with inoculating a petri plate in order to isolate the species from a body sample. Students will use tryptic soy agar plates (college-provided) and cotton swabs (student-provided) to practice inoculating (=placing the sample on the plate) body samples collected using a cotton swab. ABOUT THE PETRI PLATE: Petri dishes are special plastic containers that in Microbiology will be made into Petri plates when sterilized agar media are poured in and allowed to solidify. They consist of a top and bottom, the bottom being where the agar medium is poured, and the top is the lid. The lid is not tight fitting to allow for easy opening and closing during the inoculation process. The lid simply sits by gravity over the plate bottom (containing the agar) to prevent things from falling on the agar surface. If that thing is a microbe, the lid protects against growth of that unwanted introduced microbe; microbes we do not intentionally put on media for inoculation are called contaminants. You could think of contaminants like weeds in your garden--they just seem to pop up, and were not put there, and are, therefore, unwanted! Plates are always stored/placed upside down—this prevents any condensation (that forms from trapped water vapor) from falling on the agar surface. Wet agar surfaces don’t allow bacteria to grow at all, or to grow well (not in the pattern intended). MATERIALS NEEDED: Petri plates, for labeling either permanent marker or tape (masking or painter’s) and pen, rubber bands or tape for securing the plate once inoculated, disposable plastic bag or plastic container (such as boxes that prepared salad mixes can come in), clean cotton swabs, container of boiling water Appropriate body samples to take (only choose these—you will have 4 plates to use so you could do two of the same samples so you have a duplicate set, or choose 4 different samples): front top tooth (incisor) outer surface, between toes, belly button, behind ear, outer nose crease, armpit (DO NOT SAMPLE DEEP INSIDE YOUR BODY like throat or inside your nose, and DO NOT SAMPLE PRIVATE BODY REGIONS!) Steps for inoculating your plates: 1. First, I suggest you use one plate to explore with before you actual take the real samples to inoculate the remaining plates. With this plate, just check it out—figure which side is the top vs. bottom, how the lid works, touch the agar surface to see the texture and firmness of it (it’s ok to do this on this one plate only, it’s your “throw away” plate. Remember that it is sterile so no need to worry about touching the inside surface! Maybe try writing on the plate (outside plastic, see below) with your permanent marker to see how that works. Once you are done exploring, wrap it up tightly and securely in a bag and dispose of it in the trash—do not ever go back to open this plate up. 2. Now, prepare to begin by gathering all of your materials for inoculating your first plate (you will inoculate them one at a time)—you need one plate, your marker or tape and pen (for label), your cotton swab container (do not remove your swab until right before you use it—WHY am I telling you this?), and your container of boiling water (it doesn’t have to be a lot of water because you are only using it to moisten your swab in order to get a good sample, but please be careful because it’s boiling water). 3. Label your plate on the bottom and near the edge (write directly on the plate with your permanent marker, or if using tape, cut a thin, long strip about 1 1/2in long X 1/4in wide). List two informational items on your label, the sample source (body part chosen) and date taken. 4. Now you are ready to gather the sample and inoculate the plate: turn the plate right side up get swab and dip into container of boiling water (do not touch the sides or bottom of container)—you just want to moisten the swab so barely dip it in and when you pull it out, hold it a couple inches from the water surface to let it cool a bit (15 sec) take the body sample by zig-zagging back and forth on the area to transfer the sample on to the swab (you may spin the swab periodically to get a better sample) Immediately inoculate the plate as follows— • tilt the plate lid up in order to touch the swab to the agar surface [“pop” the lid up at an angle only (not completely vertical), but do NOT remove the whole lid or open it all the way or you will likely get contamination] • swab back and forth (zig-zag) on the top 1/3 of the plates (agar) surface—you should overlap your zig- zags and periodically spin the swab to ensure good transfer of collected sample • on the remaining 2/3 of the plate do the following—using the same swab, pull down from the area you just inoculated straight down into the remaining 2/3 of the plate so that you get several lines you pull down perpendicular to your initial zig-zag inoculation (see drawing) • place the lid down over the plate fully, dispose of your swab in the trash, and secure your plate so it cannot open accidentally by either binding it around the middle with a rubber band, or using some tape on the edges—this plate should never be opened, accidentally or intentionally • do the same process with your other plates and different body samples • when all done, incubate your plates in a bag and in a place where it is not too cold or hot. check for growth after 2 days and keep checking everyday after that up to one week and one day. When your growth (roundish dots called colonies) are at least about the size of a sesame seed, you are ready to complete the assigned questions. After you decide they are ready to evaluate, do the following below and then discard as directed below. Do not keep your plates past their use and do not put them in the fridge after inoculation. This is a concentrated sample. Do not allow your pets or children near these! Read on, for what to turn in for your assignment. OBSERVATIONS: How long did growth take to appear? Is the growth typical of bacteria (smooth and paste-like) or mold (hairy and filamentous), or do you see both? if you grew mold, try to ignore it since it is not from your body! Focusing on the bacterial colonies only, based on differences you see in relation to colony size, color, shape, surface attributes (like smooth, shiny, wrinkles, for example), determine the number of different species you have and record here. Do you have good isolation of the different species in your sample (colonies of the different types that are not touching)? You may have some species that are well-isolated while other are not, please note any differences you see. Using the lab manual to identify the correct terminology for colony description, describe one wellisolated colony (or, if you didn’t get good isolation then note the color and surface features only). Write NA if you did not get isolated colonies. IF you didn’t get growth on some or all of your plates, explain what you think are the possible reasons why you didn’t get growth. Tell us about your overall experience— Were you successful? (how do you know?) Were there any things you would do differently? Did you make mistakes? Did your agar crack? Did you make improvements after the first plate you inoculated--what adjustments did you make? Did you follow the steps in the right order, or did you forget anything? Please share any final thoughts about this experience of streak plating. You must include, along with this completed document, pictures of your labeled plates (your labels must be clear and visible in the pictures) BEFORE incubation (growing them). You must include pictures of those same plates (your labels must be clear and visible in the pictures) AFTER incubation (growing them). Make sure that the growth is visible in the pictures if growth occurs. Include pictures of ALL plates, even the ones that did not grow any microbes. PROPER PLATE DISPOSAL—once you make your observations and take your photographs, wrap up your plates so they cannot be opened, put in your ziplock bag and place in another bag and tape that bag shut. Place it in a secure outdoor trash bin so that no person or animal can get into them! Figures from https://greenbioresearch.com/culture-bacteria-using-pre-made-nutrient-agar-plates/ beteren Foes 5120121 ear 5120125 behind Cour 512012) behind 5120 121 2 an 5120 121 2 an bellecen Toes 520 boulton 5120129 Belly beteren Foes 5120121 ear 5120125 behind с A filamentos 2 B 5 mm in diameter. In addition to being filamentous, they are convex, 2.13 Filamentous Colonies - (A) These colonies of Rhizobium leguminosarum were grown on brain-heart infusion agar and are about circular , and mucoid with translucent edges. R. leguminosarum is capable of causing root nodule formation (rhiz means "root") in many legumes and subsequently fixing atmospheric nitrogen. (B) This micrograph of three R. leguminosarum colonies on brain-heart infusion agar was taken through a stereo microscope. (C) Fungi other than yeasts grow as filaments called hyphae, which collectively are referred to as a mycelium. Shown is an unidentified mold mycelium obtained from the microbiology lab viewed with a stereo microscope. Common Colony Margins (Figures 2.14-2.18) 2.14 Entire or Smooth Margin These circular and convex Rhodococcus rhodochrous colonies were grown on brain-heart infusion agar for 48 hours. They are about 1 mm in diameter with a smooth margin and a shiny, pink surface (hence "rhodo"). Rhodococcus species are soil organisms. 2.16 Spreading Margin - Erwinia amylovora colonies show an irregular spreading margin. Notice that not all colonies exhibit this feature (arrow). These colonies were grown for 24 hours on tryptic soy agar at 25°C. E. amylovora is a plant pathogen. Its scientific name literally means "Erwin (Smith's) starch devourer. 2.15 Lobed Margin- This lab contaminant colony has a lobed margin and a rugose surface. Notice that even though the margin is lobed, the overall colony shape is circular. It was approximately 1 cm in diameter at 48 hours of incubation at 35°C. 2.17 Filamentous Margin Viewed with the stereo microscope, the filamentous margin of this unidentified lab contaminant is visible. The colony is also circular, convex, shiny, and about 2 mm in diameter. MICROBIOLOGY: Laboratory Theory & Application, Brief B 2 2.18 Rhizoid Margins (A) These irregular colonies of Clostridium sporogenes were grown anaerobically on sheep blood agar and are viewed through a stereo microscope. They have a raised center and a flat, spreading edge of branched, tangled filaments (reminiscent of the mythological creature Medusa, who had snakes for hair!). They vary in size from 2 mm to 6 mm. C. sporogenes is found in soils worldwide. (B) Fungi (other than yeast) see that the margin is rhizoid and not simply filamentous. The black spheres are asexual spores called conidia. Colony Textures (Figures 2.19-2.22) A B 2.19 Mucoid Colonies - (A) These Klebsiella pneumoniae colonies grown on nutrient agar are mucoid, raised, and shiny. While it is a normal inhabitant of the human intestinal tract, it is associated with community-acquired pneumonia and nosocomial urinary tract infections. (B) Pseudomonas aeruginosa grown on Endo agar illustrates a mucoid texture. P. aeruginosa is found in soil and water and can cause infections in burn patients. es 2.20 Butyrous Colony - This unidentified 12 mm colony was found on a glycerol yeast extract plate inoculated with a diluted soil sample. Butyrous (butyrum means "buttery") colonies have the consistency of melted butter. This colony was almost liquid in texture, something that is demonstrated by its contact with the yellow colony to its right. 2.21 Granular Colony - These colonies of Streptomyces grown on brain-heart infusion agar are circular, entire, and gra a ridged surface. At a later stage of development, they produc reproductive spores. Growth of streptomycetes is associated "earthy" smell. This one plate fragranced the entire incubator SECTION 2 Microbial Growth ca growth pattern that resembles fungi (but they are Gram-positive 2.22 Dry Colony Shown are colonies of Streptomyces violaceus viewed with the stereo microscope after 3 weeks of incubation at 25°C. They are umbonate, granular, and dry Members of this genus share a bacteria and the similarities are only coincidental) and some fungal terminology has traditionally been used to describe them. For instance, their filaments are called hyphae and the colony is called a mycelium. They also grow aerial hyphae that produce chains of reproductive spores (different from bacterial endospores). These colonies were about 4 mm in diameter and adhered tenaciously to the agar surface. 2 Optical Properties of Colonies (Figure 2.23) B B A Subs Obique 2.23 Opaque and Translucent Colonies - (A) These colonies were photographed with a stereo microscope using reflected light. There are slight differences in size and color, and one is irregular in shape, whereas the others are circular. But there is another difference not visible with reflected light. (B) Here, the same colonies were photographed with transmitted light and it becomes obvious that the medium-sized round colonies are translucent, whereas the others are opaque. Other Less Common, But Distinctive, Colony Features (Figures 2.24-2.25) affuced Stews vulgaris notild 24 swarming Growth Members of the genus Proteus will rm at certain intervals and produce a pattern of concentric rings use of their motility. This photograph demonstrates the swarming vior of P. vulgaris on DNase agar. 2.25 Diffusible Pigment The blue-green pigment pyocyanin diffusing from the growth is distinctive of Pseudomonas aeruginosa. Here P.aeruginosa is growing on tryptic soy agar. MICROBIOLOGY: Laboratory Theory & Annlin Species Diversity (Figure 2.26) dry A B 2 C D iman 2.26 Comparison of Four Bacillus Species Colonies . (A) B. cereus grown on sheep blood agar (SBA) produces distinctively large (up to 7 mm) , gray, granular , irregular colonies. They often produce a "mousy" smell. Also note the distinctive extensions of growth along the streak line. (B) B. anthracis colonies on SBA resemble B. cereus, but are usually smaller and adhere to the medium more tenaciously. It must be handled at least in a BSL-2 laboratory, and sometimes BSL-3 if the cell density is high enough, so it is unlikely you will be testing its tenacity! (C) B. mycoides produces distinctive, rapidly spreading, rhizoid (note the branching) colonies. It is a common isolate from soil and is shown here on SBA. (D) This unknown Bacillus isolated as a laboratory contaminant produced a wrinkled, irregular colony with an irregular (wavy) margin on tryptic soy agar. Filaments Rhizoid dry Extrinsic Factors Affecting Colony Morphology (Figures 2.27-2.30) B A 2.27 Effect of Incubation Time on Colony Morphology (A) Close-up of Bacillus subtilis on sheep blood agar after 24 hours of incu (B) Close-up of the same Bacillus subtilis culture after 48 hours of growth. Note the wormlike extensions. SECTION 2 Microbial Growt Luiz #2 of m 177- s 18 Cage AD after 48 hours 28 howo B A. 2 Effect of Incubation Time on Pigment of S. marcescens after 48 hours. Note in particular the change in the three colonies in the lower right (circled). (A) Serratia marcescens grown on nutrient agar after 24 hours. (B) The same plate 30< grow 317 kose pegedett 37 25 2.29 Effect of Incubation Temperature on Pigment Production - Pigment production may be influenced by temperature. Serratia marcescens produces less orange pigment when grown at 37°C (left) than when grown at 25°C (right). Migh Ten will LOSS Rigmention 2.30 Effect of Nutrient Availability on Pigment Production Pigment production may be influenced by environmental factors such as nutrient availability. Chromobacterium violaceum produces a much more intense purple pigment when grown on tryptic soy agar (left) than when grown on nutrient agar (right), a less nutritious medium. turbid al GoM References Claus, G. William. Chap. 14 in Understanding Microbes—A Laboratory Textbook for Microbiology. New York: W. H. Freeman and Co., 1989. Collins, C. H., Patricia M. Lyne, and J. M. Grange. Chap. 6 in Collins and Lyne's Microbiological Methods, 7th ed. Oxford, England: Butterworth-Heinemann, 1995. Tille, Patricia M. Pages 92-93 in Bailey & Scott's Diagnostic Microbiology, 13th ed. St. Louis, MO: Mosby, 2014. Winn, Washington C. et al. Koneman's Color Atlas and Textbook of Diagnostic Microbiology, 6th ed. Baltimore: Lippincott Williams & Wilkins, 2006. MICROBIOLOGY: Laboratory Theory & Application, Brief EXERCISE Colony Morphology 2-2 Theory Odou be describe based on cherat. solid nutrient medium, it begins to divide. One cell makes When a single bacterial cell is deposited on an appropriate .. two, two make four, four make eight two million, and so on. Eventually a visible mass of cells a colony-appears where the original cell was deposited. Color, size, shape, and texture of microbial growth are determined by the genetic makeup of the organism (in greatly influenced by environmental factors, including Colony morphological characteristics may be viewed inpoint of Pun " cribe tiny, pinpoint). Elevations are best viewed with the plate tilted slightly 2 at eye level. Opacity and translucence are best viewed by one million make placing the plate on a colony counter or holding it (lid on) so it is illuminated from behind (transmitted light). Colony dimensions are best measured from the plate's base rather than through the lid. When reporting colony morphology, it is important many cases by yet unknown mechanisms), but are also to include the medium and the incubation time and temperature, all of which can affect a colony's appearance. nutrient availability, temperature, and incubation time, with the naked eye, a hand lens, a stereo (dissecting) Application microscope, or a colony counter (Fig. 2.3). The seven basic Recognizing different bacterial growth morphologies on agar plates is a useful step in the identification process. It is categories include colony size, shape, margin (edge), surface, elevation, texture, and optical properties (Fig. 2.4). often the first indication that one organism is different from 1. Size is simply a measurement of the colony's another. Once purity of a colony has been confirmed by ar appropriate staining procedure (this is not always done dimensions—the diameter if circular or length cells can be transferred to a sterile medium, grown, and and width if shaped otherwise.Pa head maintained as a pure culture, which then acts as a sour ), of that microbe for identification or other purposes. 3. The margin may be entire (smooth, with no irregularities), undulate (wavy), lobate (lobed), filamentous (unbranched strands), or rhizoid (branched like roots). 4. The surface may be smooth, rough, wrinkled (rugose), shiny, or dull. 5. The texture may be moist, mucoid (sticky), butyrous (buttery), or dry. 6. Elevations include flat, raised, convex, pulvinate (very convex), and umbonate (raised in the center). 7. Other useful features include color and optical properties such as opaque (you can't see through it) A beige and translucent (light passes through). Features such as colony shape, margin, surface, QUEBEC Darkfield Colony Counter Reichert Shape of texture (shiny or dull), and color are best viewed by observing from above while holding the plate level with the lid off (if it is safe to do so), but rocking it back and forth slightly so reflected light hits it at different angles. If allowed to do so, you may also check texture by touching the growth with an inoculating loop or wooden stick. Be sure to flame the loop afterward or dispose of the wooden stick properly. 2.3 Colony Counter Subtle differences in colony sha can best be viewed with magnification, such as is provided counter. The transmitted light and magnifying glass allow of greater detail; however, colony color and many other fe best determined with reflected light. The grid in the back counting aid; each big square is 1 square centimeter. Pnpoint strepto cocci Cali Bacillus muald 191 colony diameters (in mm) with a ruler and include In This Exercise Today you will be viewing colony characteristics on the plates saved from Exercise 2-1 and (if available) prepared streak plates provided by your instructor. Figures 2.4 through 2.30 show a variety of bacterial colony forms and characteristics. Where applicable, contrasting 2 environmental factors are indicated. them with your descriptions in the table on the data sheet, page 77. If you see a distinctive feature that has not been given a name, make up one! Just make it descriptive and easily understood by others. That's what the early microbiologists did to compile the list you have been given. (Note: Remember that many microorganisms are opportunistic pathogens, so be sure to handle the plates carefully. Do not open plates with BSL-2 organisms on them or those containing fuzzy growth, because a fuzzy appearance suggests fungal growth containing spores that can spread easily and contaminate the laboratory and other cultures. If you are in doubt, check with your instructor.) 2 Unless you have been instructed to save today's cultures for future exercises (such as Exercise 2-3), discard all plates in an appropriate autoclave container. ) Materials Per Student Lab coat Disposable gloves Chemical eye protection Whole Colony Irregular Spindle Filamentous Rhizoid Round cercumference Per Student Group (Optional) colony counter, stereo (dissecting) microscope, or hand lens Metric ruler Plates from Exercise 2-1 (Optional) tryptic soy agar or brain-heart infusion agar streak plate cultures of any of the following: · Bacillus subtilis · Corynebacterium xerosis · Kocuria rosea · Lactobacillus plantarum or Lactobacillus acidophilus • Micrococcus luteus • (Optional) tryptic soy agar or brain-heart infusion agar streak plate cultures of these BSL-2 organisms: • Mycobacterium smegmatis (BSL-2) • Proteus mirabilis (BSL-2) Margin Lobate Smooth, entire Irregular (erose) Filamentous Rhizoid (Filiform) Elevation Convex Umbonate Flat Plateau PROCEDURE 1 Working with your group, use the terms in Figure 2.4 and in the text to describe some representative colonies on your plates from Exercise 2-1 (if not already described) and the pure cultures supplied. Figures 2.5 through 2.30 may also be useful. Measure Raised Raised, Flat, raised spreading margin Growth into medium edge 2.4 A Sampling of Bacterial Colony Features These terms are used to describe colony morphology. Descriptions also should include color, size, surface characteristics, texture, and optical properties (opaque or translucent). See the text for details. Diversity of Colony Morphologies in Mixed Cultures (Figures 2.5-2.6) 2 2.5 Two Mixed Soil Cultures on Nutrient Agar These plates show the morphological diversity present in two diluted soil samples incubated for 48 hours. If two colonies look different when grown under the same conditions, they most likely are different species. The opposite is not always true, however. Two different species can produce colonies that are virtually identical. B A 1 C 2.6 Throat Cultures Grown on Sheep Blood Agar (A) There are probably five different species in this portion of th plate. (B) Note the a-hemolysis (darkening of the agar) shown b much of the growth.a-hemolytic organisms are abundant in thr samples and the majority are harmless commensals. (C) This is close-up of the boxed area in B. Note the weak B-hemolysis (cle of the agar) by the white colony in the upper right (arrow). W growth with B-hemolysis is characteristic of Staphylococcus For information about hemolytic reactions on blood agarp see Exercise 5-21. Common Colony Morphologies and Colors (Figures 2.7-2.13) B A 2 2.7 Round, Shiny, Convex Colonies (A) These buff-colored colonies of Providencia stuartii grew.on nutrient agar in 48 hours. P. stuartii is a frequent isolate in urine samples obtained from hospitalized and catheterized patients. It is highly resistant to antibiotics. (B) The colonies of the soil and water bacterium Chromobacterium violaceum grown on sheep blood agar are purple (hence "violaceum”). The effect of nutrient availability on pigment production by C. violaceum is shown in Figure 2.30. B A 2.8 Round, Dull, Convex Colonies - (A) Dry, buff-colored Corynebacterium xerosis colonies on sheep blood agar photographed from the side C. xerosis is rarely an opportunistic pathogen. (B) Close-up of the same C. xerosis colonies, but from above. 2.9 Irregular Colony Shape - This unidentified contaminant grew on tryptic soy agar and was isolated from a laboratory tabletop. In addition to its irregular shape, it has a lobed margin and wrinkled (rugose) surface. Its widest dimension was approximately 5 mm. This photo was taken through a stereo microscope. 70 MICRORIRLOCK umbonate pinpoint A Pund N B B 2.11 Umbonate Colonies (A) The colony on the left of this anaerobic lab contaminant is truly umbonate. The one on the right is getting there. Their diameters are about 3 mm. (B) These Enterococcus faecium colonies were grown on tryptic soy agar for 48 hours and were photographed using a stereo microscope. The colonies are 1-2 mm in diameter and are white, circular, and umbonate (note the thicker center) with an entire margin. Notice, though, how flat the colonies are except for the central bump that makes them umbonate. E. faecium (formerly known as Streptococcus faecium) is found in human and animal feces. C 2.10 Punctiform Colonies (A) Shown are Mycobacterium negmatis colonies grown on sheep blood agar. The colonies of this ow-growing relative of M. tuberculosis are less than 1 mm in diameter. These punctiform colonies of the rose-colored Kocuria rosea are 5 mm in diameter and were grown for 96 hours. (C) These are the me colonies as in (B) but viewed with a stereoscopic microscope. tice their irregular shape and wrinkled surface. K. rosea is an abitant of water, dust, and salty foods. 2.12 Flat Colony - This unknown soil isolate is flat only around the edge. The center is concave (but mostly flat, not concave like a bow. and at the very center is another small, raised ring surrounding anothe concavity. It's almost like an automobile tire around the wheel and hu Very unusual! Also note the dull surface. SECTION 2 Microbial Growth e
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