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      • VOLCANO LAB
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    • MIDTERM 2 STUDY GUIDE
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      • Midterm 3 Study Guide Population Ecology
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  • Environmental Biology Laboratory
    • SOILS AND GROUNDWATER
    • Ecological Roles of Living Organisms
      • The Basics
      • Bacteria - Ecological Roles
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    • Virtual FIELD TRIP TO THE RIO HONDO COLLEGE ​WILDLIFE SANCTUARY - Adaptations to Dry Climates
    • Microscopic Plant Adaptations
    • Natural Selection
    • GROWTH CURVES
    • SOILS AND GROUNDWATER
    • LC50 and LD50
    • How to Make a Solar Water Heater
    • WATER QUALITY ANALYSIS
  • General Biology
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    • Introduction to The Cell
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    • GENERAL BIOLOGY 101 LABORATORY HOME PAGE
      • Enzymes
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      • Lab 1 - Bacteria, Protista and Fungi
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    • GENERAL BIO 1110L Labs
      • lab 2 - CELLS - BIO 111L
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      • lab 4 - The Circulatory System - BIO 111L
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    • Cells
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  • Human Biology Lab
    • Testing for Sugar, Starch and Proteins
    • Osmosis, Diffusion and Filtration
    • buffers
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    • Lesson 1 - Introduction to Human Sexuality
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    • Midterm Exam Study Guide
    • Lesson 6 - Fetal Development and Sexual Differentiation
    • Lesson 7 - Disorders of Sexual Development
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    • aseptic technique
    • WET MOUNT
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    • Blood Agar
    • Dilution Series and Calculations
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    • MICROBIOLOGY UNKNOWN LAB
    • Microbiology Lab -study guide exam one
    • Ex 2 - Microorganisms
    • EX 3 - aseptic technique
    • Ex 4 - Smear Prep
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    • Verne the Sperm and friends
      • Verne the Sperm pg1
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  • Lab 6 - The Chemistry of Cells
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    • Anatomy Basics
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    • Cells Portal
      • Anatomy of the Cell SAC
      • Membrane Transport
      • The Cell Cycle
      • REGULATION of The Cell Cycle
      • BLOOD CELLS
      • mitosis
    • Tissues Portal SAC
      • The Integumentary System
      • Epithelial Tissues
      • Connective Tissue
      • Muscle Tissue
      • BONES AND SKELETAL TISSUES
      • Cartilage SAC
    • Organ Systems
    • Portal to the Skeletal system
      • The SKULL ANATOMY
      • the Thoracic Cage
      • the vertebral column
      • The Appendicular Skeleton
      • BONES AND SKELETAL TISSUES
      • joints
    • The Muscular System Portal
      • Muscle Tissue
      • Muscles - Intramuscular Injection Sites - WCU
      • Muscles of the Body - Review
    • The Nervous System
      • Introduction to the Nervous System
      • Nervous Tissue
      • The Brain - Anat
      • The Ear - Sensory Organs
      • The Eye - Sensory Organs
    • THE REPRODUCTIVE SYSTEM
    • The Renal System
    • The Respiratory System
    • THE CIRCULATORY SYSTEM PORTAL
      • Intro to the Circulatory System
      • THE HEART
      • HEART DISSECTION PHOTO GALLERY
      • THE VESSELS OF BLOOD CIRCULATION
    • Digestive System
    • Animal Dissection (Virtual)
    • dissection of the fetal pig
  • Physiology
    • Homeostasis - Physio
    • Chemical Reactions - Physio
    • Chemistry of Life - Inorganic - Physio
    • The Chemistry of Cells - ORGANIC - Physio
    • Chemical Bonds - Physio
    • Metabolism - Physio
    • Portal to the Skeletal system
    • Endocrine and Homeostasis physio
    • Muscle Physiology
    • Blood
    • Cardiovascular System
    • Lymphatic System
    • Respiratory System Physiology
    • Renal System
    • Digestive System
    • Reproductive System
  • CMC Physiology Lab
    • Lab 1 - Surface Area to Volume Ratios
    • Lab 2 - Osmosis
    • Lab 4 - Heart Rate and Barometers
    • Lab 5 - Virtual Neuron Lab
    • Case Study One
  • Anat & Physio
    • The Muscular System Portal
    • The Integumentary System a&p
      • The Epidermis
      • The Dermis
      • The Epidermis rio
      • Connective Tissue
  • Biology of Human Pregnancy
    • Course Calendar - BIO 3070
    • Bio of Pregnancy - SYLLABUS
    • Course Information
    • Evolution of Human Pregnancy
    • History of Human Pregnancy
    • Myths of Pregnancy and Fertility
    • Female Reproductive System
    • The Menstrual Cycle
    • The Male Reproductive System and Male Contraception
    • Fertility and Conception
    • In-Vitro Fertilization
    • Infertility
    • Genetics of Reproduction
    • Prenatal and Maternity Care
    • The Pregnant Body
    • fetal development
    • Development of the Nervous System
    • Stages of Labor
    • Postpartum Issues
    • Twins
  • Chemistry
    • pH Lab
    • The Chemistry of Cells - ORGANIC
      • VOLCANO LAB
    • Volcano Project
  • College/Life Skills
    • Online Professionalism
    • Advising Resources
    • INTERVIEW SKILLS AND RESUME WRITING
    • DIVERSITY
    • CAMPUS EVENTS
      • Predation
    • Time Management
  • Environmental Science
    • MIDTERM 2 STUDY GUIDE
    • Exam 2 Study Guide
    • ENVS 105 Home Page
      • Midterm 3 Study Guide Population Ecology
      • Ecology II - Communities and Ecosystems
      • Module 1 Assignments
      • Module 2 Assignments
    • Inrtoduction to ENV SCI
    • Historical Perspective of ​Environmental Science
    • Biomes
    • FOOD CHAIN and FOOD WEB
    • Biogeochemical Recycling
    • Evolution - Our Beginning
    • Genetic Inheritance
    • Evolution: How Populations Change over Time
    • Symbiosis
    • Population Ecology
    • Competition in Nature
    • Herbivory
    • Niches
    • Fossil Fuels
  • Environmental Biology Laboratory
    • SOILS AND GROUNDWATER
    • Ecological Roles of Living Organisms
      • The Basics
      • Bacteria - Ecological Roles
      • Protists - Ecological Roles
      • Fungus - Ecological Roles
      • Plantae and Animalia - Ecological Roles
    • Virtual FIELD TRIP TO THE RIO HONDO COLLEGE ​WILDLIFE SANCTUARY - Adaptations to Dry Climates
    • Microscopic Plant Adaptations
    • Natural Selection
    • GROWTH CURVES
    • SOILS AND GROUNDWATER
    • LC50 and LD50
    • How to Make a Solar Water Heater
    • WATER QUALITY ANALYSIS
  • General Biology
    • Characteristics of Life
    • Chemistry of Life - Inorganic
    • The Chemistry of Cells - ORGANIC
    • Introduction to The Cell
    • Photosynthesis and cellular Respiration
    • Cell Membranes and Osmosis
    • The Cell Cycle
    • REGULATION of The Cell Cycle
    • Mitosis
    • Meiosis
    • The Structure of DNA
    • Evolution
  • General Biology Laboratory
    • GENERAL BIOLOGY 101 LABORATORY HOME PAGE
      • Enzymes
      • OSMOSIS LAB
      • Lab 1 - Bacteria, Protista and Fungi
      • Lab 2 - Plantae and Animalia
      • Photosynthesis
      • Lab 5 - Introduction to Cells
      • Lab 6 - The Chemistry of Cells
      • Lab 7 - Membrane Transport
      • Lab 8 - Enzymes
      • Lab 9 - Photosynthesis
      • Lab 10 Fermentation, Aerobic Cellular Respiration and Associated Major Organ Systems
    • GENERAL BIO 1110L Labs
      • lab 2 - CELLS - BIO 111L
      • lab 3 - DIFFUSION and OSMOSIS - BIO 111L
      • lab 4 - The Circulatory System - BIO 111L
      • lab 6 - Photosynthesis and Cellular Respiration
      • lab 7 - Reproduction - BIO 111L
      • DNA, GENES AND GENETIC INHERITANCE
      • lab 9 - GENE EXPRESSION AND PROTEIN SYNTHESIS
      • lab 10 - ADAPTATIONS - BIO 111L
      • lab 11 - ECOSYSTEMS AND BIODIVERSITY
  • Human Biology
    • A History of Human Biology
    • Levels of Organization
    • The Chemistry of Cells - ORGANIC
    • Cells
    • Cartilage SAC
    • BONES AND SKELETAL TISSUES
  • Human Biology Lab
    • Testing for Sugar, Starch and Proteins
    • Osmosis, Diffusion and Filtration
    • buffers
    • OSMOSIS LAB
    • Anatomical Planes
    • Body Cavities and Membranes
    • Anatomical Positions
    • The Appendicular Skeleton
    • The SKULL
    • the Thoracic Cage
    • the vertebral column
  • Human Sexuality
    • Course Information
    • Course Calendar
    • Lesson 1 - Introduction to Human Sexuality
    • Lesson 2 - Genetic Inheritance of Human Sexuality
    • Lesson 3 - The Male Reproductive Tract
    • Lesson 4 - The Female Reproductive Tract
    • Lesson 5 - The Menstrual Cycle
    • Midterm Exam Study Guide
    • Lesson 6 - Fetal Development and Sexual Differentiation
    • Lesson 7 - Disorders of Sexual Development
    • Lesson 8 - Gender Identity and Sexual Attraction
    • Lesson 9 - Fetishism
    • Lesson 10 - Sexuality Throughout the World
    • ​Lesson 11 - Sexuality Through the Ages
    • Lesson 12 - Sexual Harassment, Coercion and Violence
    • Final Exam Study Guide
  • Microbiology PORTAL
    • Microbiology - CPP
      • ​Intro to Microorganisms
      • Diseases
      • EPIDEMIOLOGY
      • HOST DEFENSES
      • PATHOGENICITY
      • History of Microbiology
      • Levels of Organization cpp
      • Bacteria versus Archaea
      • Intro. to Bacteria
      • Viruses and Prions
      • Microbial Genetics
      • Microbial Nutrition and Growth
        • Nutritional Categories
        • Microbial Metabolism
        • CONTROL OF BACTERIA GROWTH AND ANTIBIOTICS
      • Eukaryotic Organisms
      • Archaeal Diversity
      • Prokaryotic and Eukaryotic Cells
      • Bacteria vs Archaeal Structures
      • Taxonomic Classifications
      • Archaea, Bacteria and Eukaryotic Cells
      • MIC- CPP Course Calendar
    • Cell Theory
    • Chemistry of Life
      • Chemical Bonds
      • Chemical Reactions
    • Biofilms
    • Definition of Terms
  • Microbiology Laboratory
    • Cell Culture and Inoculations
    • aseptic technique
    • WET MOUNT
    • Streak Plate
    • Mannitol salt agar (MSA) Test
    • Eosin Methylene Blue (EMB)
    • Blood Agar
    • Dilution Series and Calculations
    • Phage Plaque Assay
    • MICROBIOLOGY UNKNOWN LAB
    • Microbiology Lab -study guide exam one
    • Ex 2 - Microorganisms
    • EX 3 - aseptic technique
    • Ex 4 - Smear Prep
    • Ex 5 - Simple Stains
    • Ex 6 - Negative Staining
    • Ex 8 - Gram Stain
    • Ex 9 - Acid-Fast Stain
    • Ex 10 - Endospore Stain
    • Ex 11 - Motility Test
    • ex 12 -​ Pure culture technique
    • ex 13 - UV Radiation
    • Ex 14 - Enumeration of Bacteria : Standard Plate Count
    • ex - 15 Effects of Temperature on Growth
    • ex 16 - Hand-washing
    • ex 17 - pH and microbial growth
    • ex 18 - Evaluation of Antiseptics
    • ex 19 - Antibiotic Sensitivity : Kirby-Bauer Method
  • HISTOTECHNOLOGY
  • The Brain
  • The Brain
  • The Structure of DNA
  • Contact
  • FUN ZONE
    • GAMES
    • Video Vault
    • Population Ecology - ACTIVITY
    • The Carbon Cycle - ACTIVITY
    • Evolution - ACTIVITY
    • The Cell Game
    • SYMBIOSIS ACTIVITY
    • THE LORAX ACTIVITY
    • Brittney the Kidney
    • From Soup to Poop
    • MITOSIS - THE NURSERY RHYME
    • Verne the Sperm and friends
      • Verne the Sperm pg1
        • Verne the Sperm pg2
        • Verne the Sperm pg3
        • Verne the Sperm pg4
        • Verne the Sperm pg5
  • Lab 6 - The Chemistry of Cells
  • A History of Anatomy
  • List of Pages
    • Microscopes
  • Cell Membranes and Osmosis
  • Chemistry of Life
  • Muscle Movements
  • The Muscles of the Head, Trunk and Shoulders
  • The Muscles of the Limbs
  • Nervous Tissue
  • The Brain - Anat and Physiology
  • Instructions for Taking BIO 3070
  • MTH 121 Algebra A - Course Schedule and Info
  • Laboratory Calendar CMC Spring 2019
  • Genetics Lab
  • Chemistry and Conversions Lab
  • Digestion and Enzymes Lab
  • Endocrine and Homeostasis Lab
  • Muscles and Reflexes Lab
  • Sensory Lab
  • Immunohistochemistry
  • Blood Lab
  • Heart Rate, Blood Pressure, Electrocardiogram Lab
  • Respiratory Lab
  • Lab 11 Renal Lab
  • Blood Typing Game
  • Body Systems Interactive
  • Ch 9 - The Central Nervous System
  • Ch 10 - Sensory Systems
  • Neuron Virtual Laboratory
  • Virtual Eye Lab
  • Virtual pH Lab
  • Chemical Bonds Virtual Lab
  • Beer's Law Virtual Lab
  • Build-an-Atom Virtual Lab
  • Diffusion Virtual Lab
  • Ohm's Law Virtual Lab
  • New Page
  • Ch 8 - Nervous System

Aseptic Technique

Experiment 3: Aseptic Technique

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   What is a "cell culture"? The definition of a "cell culture" is the process of growing cells (or cell colonies) under controlled conditions for scientific purposes. This is almost always performed in a lab, or in some other sort of environment, other than the natural environment of the microorganism. 
   When we study cells, it is convenient to have a lot of them to study. We can add just a few selected cells of interest into a medium with all of the yummy nutrients they need to survive and reproduce. Since microorganisms reproduce using binary fission, we end up with a lot of identical cells. 
Picture
   Microbiologists use the term growth to indicate an increase in a population of microbes rather than an increase in size. One of the reasons prokaryotic organisms, like bacteria, have been so successful for billions of years, is that they reproduce very quickly using a process called binary fission. Binary fission creates two genetically identical daughter cells from one single parent cell. This process is more simple than mitosis, which occurs in eukaryotic cells, and it requires less energy. 

   What you need to know -->  Cells are really small. When we want to study them, we are not going to take one single cell and view it on a slide or run experiments on it... that would make no sense. How would you even keep track of just one cell! Instead, microbiologists will select the cell (or group of cells) that they are interested in, and they will grow them in culture
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Growth, in terms of microbiology, refers to the number of individual organisms that make up a population of microbes. For example, when bacteria cells are allowed to grow on an agar plate, one single bacterial cell will reproduce via binary fission and give rise to a colony or genetically identical bacteria cells that were all generated from a single parent cell.

​Culturing Microorganisms  

Picture
The photo is of individual colonies of E. Coli cells grown on an agar plate.
  Microbiologists collect a small sample of microbes from a given source (we call this small sample an "inoculum") and place them into a medium that contains the nutrients needed for growth. This process is called "inoculation". This creates a cell culture.
​   The medium can be in a liquid form (called broth) or in a somewhat solid, gelatin-like form (called agar/agarose).    
Picture

Growth Media

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LB Broth Preparation for Autoclaving
Picture
Pour Warm Broth + Agar Mixture into Petri dishes or Culture Tubes
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AGAR FACTS:
  • Agar is a polymer made up of subunits of the sugar galactose (milk sugar).
  • Agar is also a component of the cell walls of several species of red algae.
  • Agar can be used in the culinary arts to thicken soups and sauces. 
  • Agar forms a firm gel at room temperature.
  • Agar melts at approximately 85°C
  • The difference in temperature from agar's melting point to the temperature at which it solidifies, 32-40°C. This is very large and make agar easy to use in the lab for preparing multiple culture plates or tubes at a time. 

   The most popular growth media is a nutrient-rich liquid known as LB Broth (Lysogeny broth). Some cells are able to grown free-floating in this type of media. However, most cells require some sort of surface to adhere to. For these cell, LB broth is mixed with a substance called "agar". The agar does not provide any nutrition, but it form a surface for cellular growth. Agar is added to the broth and heated in an autoclave for sterilization. When the mixture is cool enough to handle, yet hot enough to remain in liquid form, the agar can be poured into Petri dishes, culture plates, culture tubes, or test tubes. As the agar comes to room temperature, it will solidify into a firm solid/gelatinous material that has the nutrients from the LB broth. 

Picture
Liquid Growth Media
    Microbial growth using an agar plate will yield individual colonies of cells that have been derived from a single parent cell (ideally). This method is used in clinical laboratories to test samples of feces, saliva, cerebrospinal fluid, or blood for the presence of microorganisms. ​
  •    Requirements for growth vary depending on the type of cell. There are a lot of different types of media available for this purpose. In general, a growth media should be in a liquid (broth) form or a semi-solid (agar plate) form. The media should contain the following essential nutrients
    • amino acids
    • carbohydrates
    • vitamins
    • minerals
    • growth factors
    • hormones
    • CO2 or O2
    • pH buffer to maintain relatively neutral pH
    • osmotic pressure
    • temperature (typically 37 degrees C)
Picture
Picture
The Agar Slant
PictureNutrient Broth and Slant Culture
     The agar slant, is simply a test tube in which the warm liquefied agar was poured, then the test tube was oriented in a slanted position (approximatetely 45 degree angle) and the agar was allow to cool and solidify.
    When inoculating an agar slant, draw the loop containing the inoculum very lightly over the surface in a zigzag formation while being careful not to break the surface. A needle can be used instead of a loop to inoculate an agar slant by stabbing the needle containing the inoculum into the agar.

Aseptic Technique

    An introduction to the concepts of Aseptic Technique:
    What does "aseptic" mean? The word "aseptic" means "free from microorganisms". We use "aseptic techniques" to ensure that there is no "contamination" of unwanted or unintended microorganisms introduced into your cell cultures.

Streak Plate Procedure

Picture
"An inoculation loop, also called a smear loop, inoculation wand or microstreaker, is a simple tool used mainly by microbiologists to retrieve an inoculum from a culture of microorganisms. The loop is used in the cultivation of microbes on plates by transferring inoculum for streaking." https://en.wikipedia.org/wiki/Inoculation_loop 
Media used:
TSA – Tryptic soy agar
Agar: from red algae, melting point = 100°C, solidifies ~45°C, not a nutrient

​Organism used:

Escherichia coli – Gram (-) rod
Tryptic Soy Agar contains soy and casein which is suitable for the growth of a wide variety microorganisms. It includes organic nitrogen in the form of amino acids and polypeptides, making the medium highly nutritious.
• Disinfect your bench top surface before beginning work. You should already be doing this before you start lab and after you are done with the lab.
• Loops and needles need to be sterilized in the Bunsen burner flame till red hot. Allow these implements to cool before doing any inoculations to prevent killing the organism.
• The opening of sterile media tubes and the tube of microorganism that will be used to inoculate those sterile media tubes need to be flamed after opening and before closing.
• Flame the loop or needle after each inoculation and before a new inoculation is done. This kills any leftover organism that is still on the instrument.
• Loops are used to transfer from liquid media to liquid media or petri plates. Needles are used to transfer from solid media to other solid media or petri plates. An inoculation needle is used for retrieving solid or dense media.  An inoculation loop is used to retrieve liquid media. 
• Hold the cap of the tube with your little finger. NEVER place the cap on the bench top.
TSB – Tryptic soy broth
​

Organism used:
Escherichia coli – Gram (-) rod
​general purpose liquid enrichment medium used in qualitative procedures for the sterility test

Procedure:

BEFORE YOU BEGIN:
  • Disinfect working area (see instructions).
  • Label all sterile media with your name, media type, organism used, and lab hour. 
  • Label plates on the bottom (not the lid). DO NOT label the white part of the tube.

Transfer of E.coli from a Plate to a Sterile Slant

  1. Flame the inoculating needle until it is red hot, allow to cool.
  2. Raise the lid of the petri plate just enough to put the needle inside and remove some of the organism on the plate. Close the lid. DO NOT gouge the agar.
  3. Pick up your sterile slant with your free hand and remove the cap with your little finger that is holding the needle. Flame the opening of the tube before applying the organism on the needle to the surface of the slant. DO NOT gouge or stab the slant.
  4. Remove the needle from the tube and flame the opening before capping the tube. Flame the needle as well.
How to Transfer of E.coli from a Plate to a Sterile Slant
Transfer of E.coli from a Broth to a Sterile Plate

Transfer of E.coli from a Broth to a Sterile Plate

  1. Flame the inoculating loop or needle (your preference) until it is red hot, allow to cool.
  2. Remove the cap from the broth and flame the opening. Hold the cap with your little finger that is holding the loop/needle. Use the loop/needle to pick up organism from the broth.
  3. Flame the opening of the broth before closing.
  4. Pick up your sterile plate. Open the lid just enough to stick the loop/needle inside and glide the instrument over the surface of the agar. DO NOT gouge the agar.
  5. Cover the plate back up and flame the loop/needle.
  6. Put slants, broths, and plates into the plate rack or the tube rack at the back of the class that is labeled with the experiment name.
Next lab period
Results
Obtain both tubes and your plates. Examine them for growth and uniformity of the cultures. Ask yourself these questions.
​
  1. Were the three transfers you did successful?
  2. How can you tell that the transfers were successful?
  3. If the transfers were not successful, what may have happened?
  4. Was there any evidence of another organism (i.e. Fungus) in any of your culture tubes or on your plate? If so what may have happened?

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