Bacterial Media and Culture - Collecting Clinical ...

Bacterial Media and Culture - Collecting Clinical ...

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From the Virtual Microbiology Classroom on ScienceProfOnline.com Image: Compound microscope objectives, T. Port Laboratory Exercise 4 Bacterial Media & Culture Collecting, Culturing & Interpreting Bacterial Samples From the Virtual Microbiology Classroom on ScienceProfOnline.com Images: Liquid TSY; Clinical sample being applied to TSY agar; Arm plate; all by T. Port What am I going to learn from Lab Topic #4? Bacterial Media & Culture You will practice microbial collection techniques.

Define and use aseptic technique in microbial culture and media preparation. Use enriched, complex, selective & differential media to culture microbes. Describe colony morphology and its relationship to microbial identification. Interpret results of microbial growth on various culture media based on prepared controls. Understand the ubiquitous nature of microbes. From the Virtual Microbiology Classroom on ScienceProfOnline.com

Image: Chimp brain in a jar, Gaetan Lee Growth Media Bacteria and other microbes have particular requirements for growth. In order to successfully grow bacteria in lab, we must provide an environment suitable for growth. Growth media (singular = medium) are used to cultivate microbial growth. Media = mixtures of nutrients that the microbes need to live. Also provides a surface and the necessary moisture and pH to support microbial growth.

Before being used, media must be autoclaved, so that it is sterile (all life forms killed.) Tryptic Soy Agar (TSY) is the medium that we most often use. Complex nutrient media which supports the growth of a wide variety of microbes. From the Virtual Microbiology Classroom on ScienceProfOnline.com Image: Streak plate of E. coli, T. Port Specialized Media: McConkeys, Mannitol Salt & Blood Agar Look at the plates on your lab bench that are red/pink in color. McConkeys = lighter, purplish-pink Mannitol Salt = orangish-pink Blood Agar = very dark red Unlike TSY media, these specialized selective & differential media plates are already prepared for you.

From the Virtual Microbiology Classroom on ScienceProfOnline.com Image: McConkeys, Mannitol Salt & Blood Agar specialized media, T. Port Differential & Selective Specialized Media Q: What does selective mean? Q: What does differential mean? From the Virtual Microbiology Classroom on ScienceProfOnline.com Image: McConkeys media growing E. coli, T. Port MacConkey's (MAC) Q: Is MacConkeys selective? Explain. Q: Is MacConkeys differential? Explain.

Watch VIDEO: How to Interpret MacConkeys Agar (MAC) From the Virtual Microbiology Classroom on ScienceProfOnline.com Images clockwise from top: McConkeys agar sterile; growing E. coli, Enterobacter & Salmonella (plates clockwise from top left); Lactose fermenting and non-lactose fermenting Gram colonies on MAC. All by T. Port Mannitol Salt (MSA) Q: Is Mannitol Salt selective? Explain. Q: Is Mannitol Salt differential? Explain. Watch VIDEO: How to Interpret Mannitol Salt Agar

(MSA) From the Virtual Microbiology Classroom on ScienceProfOnline.com Images: Sterile Mannitol Salt Agar, Positive & negative differential reaction on Mannitol Salt Agar, T. Port Blood agar (BAP) Most specimens received in a clinical microbiology lab are plated onto Blood Agar. It is an enriched medium that will grow even fastidious bacteria. Also contains 5% sheep blood. This media is not selective. It is enriched and differential: Certain bacteria produce enzymes (hemolysinssay hemo-liceins) that act on red cells to produce either: * Beta hemolysis: Enzymes lyse the blood cells completely, producing a clear area around the colony. * Alpha hemolysis: Incomplete hemolysis produces a greenish discoloration around the colony. * Gamma hemolysis: No effect on the red cells. Blood agar is usually inoculated from a patients throat swab.

Watch VIDEO: How to Interpret Microbiologist are trying to detect Group A beta hemolytic Streptococcus pyogenes (a Gram-positive cocci-shaped bacteria that causes Beta hemolysis on blood agar.) Blood Agar (BAP) Normal flora of the throat will exhibit alpha or gamma hemolysis. From the Virtual Microbiology Classroom on ScienceProfOnline.com Images: Beta-hemolysis, Alpha-hemolysis and a sterile plate of Blood Agar, T. Port Watch VIDEO: How to Aseptically Po ur Bacterial Growth M edia TSY

Labeling Plates All Petri plates for this and future lab exercises should be labeled and stored in the following manner: 1. Make certain that all plates are labeled on the bottom half (i.e. the portion of the 2. You can label plastic with a sharpie; glass with a wax pencil. 3. Include the following: Petri plate that contains the media). a. b. c. 4. Your initials or identifying mark Date

Type of specimen All plates are incubated in the green storage bin (which is identified as "SAVE") in the "upside down" position. Upside down means that the of the Petri plate with media faces up. The empty of the Petri plate is down. We do not use rubber bands to hold lids in place. (Except for the plates that you may transport home) Plates will be incubated at 37 C for 24 hrs, then stored at room temperature until next week, when you will observe for results. From the Virtual Microbiology Classroom on ScienceProfOnline.com Images: Positive & negative differential reaction on Mannitol Salt Agar, T. Port Inoculate Plates With Controls You will be inoculating the four types of media we are using in this class (TSY, MAC, MSA, BAP) with four of our stock species of

bacteria (E. coli, Salmonella pullorum, Staphylococcus epidermidis, Staphylococcus aureus). Doing this will help you understand the properties of these media, and what the expected outcomes (growth, no growth, and differential color changes) look like. Q: Why do we, in a fifth section of each plate, inoculate using a sterile loop? From the Virtual Microbiology Classroom on ScienceProfOnline.com Image: Arm plate, Source unknown Normal Flora Samples 1. Arm Plate will demonstrate

the microorganism inhabiting the surface of the skin. 2. Touch Plates will demonstrate the bacteria in the epidermis of our hands. 3. Throat Swab on Blood agar. 4. Nasal Swabs on TSY & MSA. REMEMBER When placing one sample on two plates, inoculate the non-selective medium first! Very gently transfer your sample to the plate. You want to avoid gouging the surface of the media.

From the Virtual Microbiology Classroom on ScienceProfOnline.com Image: Arm plate, Source unknown Microbial Colony Morphology From the Virtual Microbiology Classroom on ScienceProfOnline.com Images: Arm plate, TSY with sample from dish washer, MacConkeys with variety of colonies, all by T. Microbial Colony Morphology Discard Bin at Back of Lab From the Virtual Microbiology Classroom on ScienceProfOnline.com Confused? Here are links to fun resources that further explain microbiology media & culture:

Smart Links Media & Culture Laboratory Main Page on the Virtual Microbiology Classroom of Science Prof Online. Germs, music by Weird Al Yankovic. Video by RevLucio. Normal Flora How to Interpret: MacConkeys (MAC), Mannitol Salt (MSA) and Blood Agar (BAP) videos from Science Prof Online. How to Pour Bacterial Growth Media into Petri Dishes, webpage, by Douglas F. Fix. Interactive page where you can select an area of the body and learn which normal flora typically colonize that location.

video from Science Prof Online. Bacterial growth Microbial Growth & Metabolism Main Page on the video and narration, YouTube, Dizzo95.. Virtual Microbiology Classroom of Science Prof Online. E. coli population growth time lapse video. (You must be in PPT slideshow view to click on links.) From the Virtual Microbiology Classroom on ScienceProfOnline.com Are microbes intimidating you? Do yourself a favor. Use the Virtual Microbiology

Classroom (VMC) ! The VMC is full of resources to help you succeed, including: practice test questions review questions study guides and learning objectives You can access the VMC by going to the Science Prof Online website www.ScienceProfOnline.com Images: Staph, Giant Microbes; Prokaryotic cell, Mariana Ruiz

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