{"id":144,"date":"2022-11-03T19:32:22","date_gmt":"2022-11-03T19:32:22","guid":{"rendered":"https:\/\/press.wpunj.edu\/microbiologylabmanual\/chapter\/15-metabolic-activities-a-inoculation-of-media\/"},"modified":"2024-05-09T13:07:55","modified_gmt":"2024-05-09T13:07:55","slug":"metabolic-activities-a-inoculation-of-media","status":"publish","type":"chapter","link":"https:\/\/press.wpunj.edu\/microbiologylabmanual\/chapter\/metabolic-activities-a-inoculation-of-media\/","title":{"raw":"Metabolic Activities A: Inoculation of Media","rendered":"Metabolic Activities A: Inoculation of Media"},"content":{"raw":"
\r\n\r\nWhen microbiologists or doctors come across a microbe that may be causing an infection, they need to be able to isolate, characterize, and identify this unknown microbe in order to treat the patient, develop further antimicrobials, or simply learn.\r\n\r\nThe process of identifying involves analyzing morphology, cultural characteristics, and physiological characteristics. Morphology<\/b> <\/b>includes viewing the cell shape, colony color, and results of specific stains, particularly Gram stain. \u201cCultural characteristics\u201d refers to how the bacteria grows on various liquid and solid media, and how it consumes oxygen (obligate aerobe vs. facultative anaerobe vs. obligate anaerobe and so on). Physiological characteristics are determined through a series of biochemical assays. A biochemical assay <\/b>is testing for aspects of microbial metabolism, which is the means of a microbe getting its energy and nutrients (carbon source) to live and reproduce. Essentially these assays, or tests, are detecting which enzymes are being producing by the bacterium. Bacteria can be differentiated by which specific enzymes are present or absent.\r\n\r\nYou will be learning the procedure of how to inoculate and perform the metabolic tests with a \u201cknown\u201d bacterium for a two-week period. You will be working in pairs. Afterwards, you will be working individually for the next two-week period identifying an unknown bacterium.\r\n\r\nFor identifying the unknown bacterial strain, you will perform a Gram stain to determine if the strain is gram positive or gram negative and observe the cell shape. You will also decide what the colony color is by examining the original culture given to you on a TSA plate. These three traits are considered most important when you are using a key to identify a stain. Unfortunately, while the colony color and other visual characteristic are very powerful, they also require extensive experience to use reliably. Only a few colony colors are easily interpreted, but when you have a distinctive color that characteristic should be given a rank just after Gram stain and cell shape. The metabolic tests are a \u201cbest-fit\u201d system which works well even if some tests fail. Not every strain of a bacterial species may produce an expected result. A positive result simply means that the metabolism of interest occurred.\u00a0 Species range from mostly positive to mostly negative results and this helps make identification possible. You have to look at all results as a whole and figure out which bacterium matches closest to a bacterium in the Key, or \u201cUnknown Booklet\u201d. The \u201cUnknown Booklet\u201d is a list of bacteria with morphological and cultural characteristics described and results of the biochemical assays. Your unknown will be one of these bacteria.\r\n\r\n \r\n\r\nMATERIALS\r\n\r\nPer<\/i> <\/i>pair<\/i> <\/i>of<\/i> <\/i>students<\/i> <\/i>for<\/i> <\/i>\u201cKnowns\u201d<\/i> <\/i>**<\/span>\r\n2 Blue racks\r\n2 Inoculating loops\r\n1 Inoculating needle\r\nWax pencil\r\n2 Bunsen Burners\r\n2 Strikers\r\n9 test tube labels\r\nCoffee can\r\n1 of each type of medium:\r\n<\/colgroup>\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n
TSA agar<\/td>\r\n(agar plate, Trypticase Soy)<\/td>\r\nNitrate broth<\/td>\r\n(yellow cap tube)<\/td>\r\n<\/tr>\r\n
Milk agar<\/td>\r\n(agar plate, opaque white)<\/td>\r\nMR-VP broth<\/td>\r\n(black cap tube)<\/td>\r\n<\/tr>\r\n
Starch agar<\/td>\r\n(agar plate, black line on lid)<\/td>\r\nPR Sucrose<\/td>\r\n(blue cap tube)<\/td>\r\n<\/tr>\r\n
Simmon's Citrate agar<\/td>\r\n(screw-top agar tube)<\/td>\r\nPR Dextrose<\/td>\r\n(green cap tube)<\/td>\r\n<\/tr>\r\n
SIM medium agar<\/td>\r\n(clear cap tube)<\/td>\r\nPR Mannitol<\/td>\r\n(red cap tube)<\/td>\r\n<\/tr>\r\n
Urea broth<\/td>\r\n(white cap tube)<\/td>\r\nPR Lactose<\/td>\r\n(yellow cap tube)<\/td>\r\n<\/tr>\r\n<\/tbody>\r\n<\/table>\r\n \r\n\r\n**Per<\/i> <\/i>student<\/i> <\/i>for<\/i> <\/i>\u201cUnknowns\u201d:\r\n<\/i><\/span>1 Blue rack\r\n1 Inoculating loop\r\n1 Inoculating needle\r\nWax pencil\r\nBunsen Burner\r\nStriker\r\n9 test tube labels\r\nCoffee can\r\n1 of each type of medium listed above\r\n\r\n \r\n\r\nPROCEDURE OF METABOLIC ACTIVITIES: INOCULATION\r\n
    \r\n \t
  1. The instructor will distribute one cultural plate per pair of students to share for the learning process (\u201cKnowns\u201d), but for identifying the Unknown Bacteria, students will work individually.<\/li>\r\n \t
  2. Inoculation of plates:\r\na.\u00a0 Label the bottom of the plates with names, date, medium name, and bacterial label (name, letter, or number).\r\nb.\u00a0 Using aseptic technique, you will inoculate the medium with a single streak of bacteria on the milk and starch plates. You will inoculate the TSA plate with two streaks of bacteria. See below. When<\/b> <\/b>you<\/b> <\/b>remove<\/b> <\/b>bacteria<\/b> <\/b>from<\/b> <\/b>streak<\/b> <\/b>plate,<\/b> <\/b>you take from an isolated colony.<\/b><\/b>\"\"\u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \"\"\r\nMilk & Starch Strike Plate\u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0TSA Strike Plate<\/li>\r\n \t
  3. Simmon\u2019s Citrate Agar (screw cap):\r\na.\u00a0 Using aseptic technique, you will transfer bacteria from an isolated colony using an inoculating loop from the bacterial plate onto a slant surface.\r\nb.\u00a0 Using a test tube label, label the tube \u201cCitrate\u201d.<\/li>\r\n \t
  4. SIM Medium (clear cap):\r\na.\u00a0 Using aseptic technique, you will transfer bacteria from an isolated colony an inoculating<\/u> <\/u>needle<\/u> from the bacterial plate, and then stab the needle all the way to the bottom on the medium.\r\nb. Using a test tube label, label the tube \u201cSIM\u201d.<\/li>\r\n \t
  5. Inoculation of all other broths (one tube of each):\r\na.\u00a0 Using aseptic technique, you will transfer bacteria from an isolated colony using an inoculating from the bacterial plate into a broth. This only has to be done once per tube.\r\nb.\u00a0 The tubes are pre-labeled by their cap-color which indicates the medium:\r\n<\/colgroup>\r\n\r\n\r\n\r\n\r\n\r\n\r\n
    MR-VP broth<\/td>\r\n(black cap broth tube)<\/td>\r\nPhenol<\/b> <\/b>Red<\/b> <\/b>(PR)<\/b> <\/b>tubes<\/b> <\/b>have<\/b> <\/b>Durham<\/b> <\/b>tubes<\/b> <\/b>inside<\/b><\/td>\r\n<\/tr>\r\n
    Nitrate broth<\/td>\r\n(yellow cap broth tube)<\/td>\r\nPR Sucrose<\/td>\r\n(blue cap broth tube)<\/td>\r\n<\/tr>\r\n
    Simmon's Citrate agar<\/td>\r\n(screw-top agar slant)<\/td>\r\nPR Dextrose<\/td>\r\n(green cap broth tube)<\/td>\r\n<\/tr>\r\n
    SIM medium agar<\/td>\r\n(clear cap agar tube)<\/td>\r\nPR Mannitol<\/td>\r\n(red cap broth tube)<\/td>\r\n<\/tr>\r\n
    Urea broth<\/td>\r\n(white cap broth tube)<\/td>\r\nPR Lactose<\/td>\r\n(yellow cap broth tube)<\/td>\r\n<\/tr>\r\n<\/tbody>\r\n<\/table>\r\n<\/li>\r\n \t
  6. Place all test tubes into a coffee can. Put lab tape onto the outside of the coffee can. Label the coffee can with your name(s), bacterial label, and date. Place in your lab section\u2019s incubator and correctly labeled shelf.<\/li>\r\n \t
  7. Store your 4 plates (this includes the original bacterial culture plate) in a rack upside down in your lab section\u2019s lab bench area at room temperature. We do not incubate the plates for a week due to dehydration of the medium.<\/li>\r\n<\/ol>\r\n[embed]https:\/\/youtu.be\/S_-af1kuW1Y[\/embed]\r\n\r\n \r\n\r\nREFERENCES\r\n\r\nBrown, A. E. (2009). Benson's Microbiological Applications: Laboratory Manual in General Microbiology. New York: McGraw Hill.\r\n\r\nChess, B. (2015). Laboratory Applications in Microbiology: A Case Study Approach. New York: McGraw Hill. Cowan, M.K. (2015). Microbiology: A Systems Approach (4th edition). New York: McGraw Hill.\r\n\r\n<\/div>","rendered":"
    \n

    When microbiologists or doctors come across a microbe that may be causing an infection, they need to be able to isolate, characterize, and identify this unknown microbe in order to treat the patient, develop further antimicrobials, or simply learn.<\/p>\n

    The process of identifying involves analyzing morphology, cultural characteristics, and physiological characteristics. Morphology<\/b> <\/b>includes viewing the cell shape, colony color, and results of specific stains, particularly Gram stain. \u201cCultural characteristics\u201d refers to how the bacteria grows on various liquid and solid media, and how it consumes oxygen (obligate aerobe vs. facultative anaerobe vs. obligate anaerobe and so on). Physiological characteristics are determined through a series of biochemical assays. A biochemical assay <\/b>is testing for aspects of microbial metabolism, which is the means of a microbe getting its energy and nutrients (carbon source) to live and reproduce. Essentially these assays, or tests, are detecting which enzymes are being producing by the bacterium. Bacteria can be differentiated by which specific enzymes are present or absent.<\/p>\n

    You will be learning the procedure of how to inoculate and perform the metabolic tests with a \u201cknown\u201d bacterium for a two-week period. You will be working in pairs. Afterwards, you will be working individually for the next two-week period identifying an unknown bacterium.<\/p>\n

    For identifying the unknown bacterial strain, you will perform a Gram stain to determine if the strain is gram positive or gram negative and observe the cell shape. You will also decide what the colony color is by examining the original culture given to you on a TSA plate. These three traits are considered most important when you are using a key to identify a stain. Unfortunately, while the colony color and other visual characteristic are very powerful, they also require extensive experience to use reliably. Only a few colony colors are easily interpreted, but when you have a distinctive color that characteristic should be given a rank just after Gram stain and cell shape. The metabolic tests are a \u201cbest-fit\u201d system which works well even if some tests fail. Not every strain of a bacterial species may produce an expected result. A positive result simply means that the metabolism of interest occurred.\u00a0 Species range from mostly positive to mostly negative results and this helps make identification possible. You have to look at all results as a whole and figure out which bacterium matches closest to a bacterium in the Key, or \u201cUnknown Booklet\u201d. The \u201cUnknown Booklet\u201d is a list of bacteria with morphological and cultural characteristics described and results of the biochemical assays. Your unknown will be one of these bacteria.<\/p>\n

     <\/p>\n

    MATERIALS<\/p>\n

    Per<\/i> <\/i>pair<\/i> <\/i>of<\/i> <\/i>students<\/i> <\/i>for<\/i> <\/i>\u201cKnowns\u201d<\/i> <\/i>**<\/span>
    \n2 Blue racks
    \n2 Inoculating loops
    \n1 Inoculating needle
    \nWax pencil
    \n2 Bunsen Burners
    \n2 Strikers
    \n9 test tube labels
    \nCoffee can
    \n1 of each type of medium:<\/p>\n\n<\/colgroup>\n\n\n\n\n\n\n\n
    TSA agar<\/td>\n(agar plate, Trypticase Soy)<\/td>\nNitrate broth<\/td>\n(yellow cap tube)<\/td>\n<\/tr>\n
    Milk agar<\/td>\n(agar plate, opaque white)<\/td>\nMR-VP broth<\/td>\n(black cap tube)<\/td>\n<\/tr>\n
    Starch agar<\/td>\n(agar plate, black line on lid)<\/td>\nPR Sucrose<\/td>\n(blue cap tube)<\/td>\n<\/tr>\n
    Simmon’s Citrate agar<\/td>\n(screw-top agar tube)<\/td>\nPR Dextrose<\/td>\n(green cap tube)<\/td>\n<\/tr>\n
    SIM medium agar<\/td>\n(clear cap tube)<\/td>\nPR Mannitol<\/td>\n(red cap tube)<\/td>\n<\/tr>\n
    Urea broth<\/td>\n(white cap tube)<\/td>\nPR Lactose<\/td>\n(yellow cap tube)<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

     <\/p>\n

    **Per<\/i> <\/i>student<\/i> <\/i>for<\/i> <\/i>\u201cUnknowns\u201d:
    \n<\/i><\/span>1 Blue rack
    \n1 Inoculating loop
    \n1 Inoculating needle
    \nWax pencil
    \nBunsen Burner
    \nStriker
    \n9 test tube labels
    \nCoffee can
    \n1 of each type of medium listed above<\/p>\n

     <\/p>\n

    PROCEDURE OF METABOLIC ACTIVITIES: INOCULATION<\/p>\n

      \n
    1. The instructor will distribute one cultural plate per pair of students to share for the learning process (\u201cKnowns\u201d), but for identifying the Unknown Bacteria, students will work individually.<\/li>\n
    2. Inoculation of plates:
      \na.\u00a0 Label the bottom of the plates with names, date, medium name, and bacterial label (name, letter, or number).
      \nb.\u00a0 Using aseptic technique, you will inoculate the medium with a single streak of bacteria on the milk and starch plates. You will inoculate the TSA plate with two streaks of bacteria. See below. When<\/b> <\/b>you<\/b> <\/b>remove<\/b> <\/b>bacteria<\/b> <\/b>from<\/b> <\/b>streak<\/b> <\/b>plate,<\/b> <\/b>you take from an isolated colony.<\/b><\/b>\"\"\u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \"\"
      \nMilk & Starch Strike Plate\u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0 \u00a0TSA Strike Plate<\/li>\n
    3. Simmon\u2019s Citrate Agar (screw cap):
      \na.\u00a0 Using aseptic technique, you will transfer bacteria from an isolated colony using an inoculating loop from the bacterial plate onto a slant surface.
      \nb.\u00a0 Using a test tube label, label the tube \u201cCitrate\u201d.<\/li>\n
    4. SIM Medium (clear cap):
      \na.\u00a0 Using aseptic technique, you will transfer bacteria from an isolated colony an inoculating<\/u> <\/u>needle<\/u> from the bacterial plate, and then stab the needle all the way to the bottom on the medium.
      \nb. Using a test tube label, label the tube \u201cSIM\u201d.<\/li>\n
    5. Inoculation of all other broths (one tube of each):
      \na.\u00a0 Using aseptic technique, you will transfer bacteria from an isolated colony using an inoculating from the bacterial plate into a broth. This only has to be done once per tube.
      \nb.\u00a0 The tubes are pre-labeled by their cap-color which indicates the medium:<\/p>\n\n<\/colgroup>\n\n\n\n\n\n\n
      MR-VP broth<\/td>\n(black cap broth tube)<\/td>\nPhenol<\/b> <\/b>Red<\/b> <\/b>(PR)<\/b> <\/b>tubes<\/b> <\/b>have<\/b> <\/b>Durham<\/b> <\/b>tubes<\/b> <\/b>inside<\/b><\/td>\n<\/tr>\n
      Nitrate broth<\/td>\n(yellow cap broth tube)<\/td>\nPR Sucrose<\/td>\n(blue cap broth tube)<\/td>\n<\/tr>\n
      Simmon’s Citrate agar<\/td>\n(screw-top agar slant)<\/td>\nPR Dextrose<\/td>\n(green cap broth tube)<\/td>\n<\/tr>\n
      SIM medium agar<\/td>\n(clear cap agar tube)<\/td>\nPR Mannitol<\/td>\n(red cap broth tube)<\/td>\n<\/tr>\n
      Urea broth<\/td>\n(white cap broth tube)<\/td>\nPR Lactose<\/td>\n(yellow cap broth tube)<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/li>\n
    6. Place all test tubes into a coffee can. Put lab tape onto the outside of the coffee can. Label the coffee can with your name(s), bacterial label, and date. Place in your lab section\u2019s incubator and correctly labeled shelf.<\/li>\n
    7. Store your 4 plates (this includes the original bacterial culture plate) in a rack upside down in your lab section\u2019s lab bench area at room temperature. We do not incubate the plates for a week due to dehydration of the medium.<\/li>\n<\/ol>\n