Menopause

Menopause
Presentation lecture from: Uni. of Maryland

"Menopause is a wake-up call to a new phase of your life. A wake-up call most people cannot handle, but what's the choice? You sure can't go back to sleep. So get up and decide what you're doing today—and on into the years ahead." —modified after Marisa Weiss, M.D.

What Is Menopause?
* Menopause is defined as the cessation of ovarian function, or the cessation of menstrual cycles
* Menopause is a natural process, it is not a disease
* The mean age for menopause is 51 years
* One third of woman’s life occurs after menopause

Menopause

* Medical
* Surgical
* “Cold turkey"
* Persistent low (less tan 20 pg/ml) levels of estrogen
* Persistent elevated levels of
FSH (13–90 milli-international units per milliliter)
LH (15–50 mIU/mL).

Normal levels of estrogen before menopause peak at 150–300 picograms per milliliter each month (depending on where you are in your menstrual cycle)

Lack of negative feedback
What Is Perimenopause?

* Perimenopause is a transition rather than an event
* It is the period of time surrounding menopause when ovarian function is declining, but has not stopped
* Onset of perimenopause is usually 3-5 years before the periods stop
* Perimenopause and menopause may last 2-10 years

Other factors then hormones:

Empty nest, divorce, widowhood, the dependency of sick parents, the death of a parent or parents, or kids in trouble are all common experiences at this stage of life. Setting aside the hormonal changes that relate to menopause, it's common to feel stress, isolation, and depression.

Menopause/Aging

"Growing old is not for sissies”
Symptoms of Perimenopause

* Hot flashes
* Vaginal dryness
* Breast Tenderness
* Mood disturbances
* Sleep disturbances
* Urinary tract infections/incontinence
* Menstrual changes
* Sexual dysfunction

Other Physiologic Changes
* Slowing of metabolism
* Weight gain
* Changes in lipids
* Increase in heart disease
* Osteoporosis

Heart Disease

* Leading cause of death in women
* Higher risk after menopause probably due to changes in cholesterol levels when estrogen levels decline
* Elevated “bad” cholesterol (LDL)
* Lower “good” cholesterol (HDL)
* Elevation in triglycerides (fats)

Osteoporosis

* 1 out of 2 white women will have an osteoporotic fracture in their lifetime
* 24% of patients over age 50 will DIE in the year following an osteoporotic hip fracture
* Deaths due to osteoporosis far exceed the numbers of deaths due to breast cancer
* Other consequences- physical limitation, chronic pain, depression, poor quality of life

Menopause

Three factors affect how you age and how you experience menopause:

* Hormones — estrogen, progesterone, and others.
* Lifestyle — diet, exercise, weight, smoking, environment
* Genetic makeup — the genes that come from your parents, the blueprint of your constitution and perhaps your future health

Estrogen Replacement Therapy (ERT) or Hormone Replacement Therapy (HRT)
* Treats the symptoms of menopause (hot flashes, etc.)
* Prevention of heart disease (40-50% reduction in risk)
* Prevention and treatment of osteoporosis
* May help prevent dementia
* May prevent strokes
* Lower mortality rates in women who take estrogen

Side Effects of HRT

* Blood clots (especially in smokers)
* Gallbladder disease
* May increase risk of breast cancer
* Vaginal bleeding
* Breast tenderness

“Male” Hormones- Can They Help You?

* Androgens are produced in the ovaries and the adrenal glands
* Known to increase libido
* Protects bone mass
* Unfavorable effects on cholesterol
* Can cause virilization (unwanted hair growth)

Complementary Therapies

* Most lack scientific proof of efficacy
* Large placebo effect
* “Dietary supplements”- no regulation by FDA
* May be helpful for women who do not choose ERT/HRT or as adjunctive therapy

Lifestyle Approaches to Perimenopause

* Exercise- aerobic, weight-bearing, strengthening program
* High fiber, low fat, soy-rich diet
* Maintain regular sexual activity
* Relaxation and stress reduction activities
* Daily sunlight
* Decrease alcohol intake
* Stop Smoking

Summary

* Perimenopause is a process rather than an event
* Symptoms are common and most women will seek treatment
* ERT/HRT is the best treatment available for symptoms of menopause and to prevent long term consequences of estrogen deficiency
* Complementary therapies may be of some short term benefit for symptom control

Menopause: Hot Flushes
Menopause: Hot Flushes Misconception
Menopause: Hot Flushes Triggers
Hot Flush Therapies
Clinical data on current possible therapies for the treatment of hot flushes (HF)
Potential Vasomotor Pathways
Thermoregulatory
Center
Hypothalamus
serotonin
norepinephrine
neuropeptides
estrogen
progesterone
autonomic
nervous system
brain stem
vasodilatation or vasoconstriction
Heat loss (i.e., hot flush) or preservation
blood vessels in skin
Morphine-Dependent OVX Model
Direct Temperature Measurement
Thermistor
Amplifier
MacLab
Telemetric Temperature Measurement
Transmitter
THERMISTOR IMPLANTED
OVX-induced change in TST
Long-term monitoring
Dark phase (active)
Light phase (inactive)
OVX-Induced TST Change
Telemetry (Long-Term Monitoring)
Hot Flush - Gonadotropins
Finger temperature
Relative hormone levels during the menstrual cycle
Aging-related decline of hormonal systems
Hormone levels of women and men during aging

Andropause
Serum testosterone levels in young vs aged men
Gonadotropins and aging

Menopause.ppt

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Microbiological Methods

Microbiological Methods

Making Media
Pouring Culture Plates
Sterile Technique
Inoculating Plates and Culture Tubes
Use of a Plate Counter to Estimate Microbial Population Densities

Culturing Microorganisms
Sterile Technique

* When culturing bacteria or other microorganisms, it is important to keep your work area as clean as possible.
* This prevents the introduction of other microorganisms from the environment into your culture.
* The techniques used to prevent contamination are referred to as sterile techniques.
* Start by washing your down your work or lab benches with a surface disinfectant. The most commonly used disinfectants for lab use are:
o 10% bleach (recommended by the CDC)
o 85% ethanol

Sterile Technique (2)

* Turn off any forced air heating or air conditioning units that create strong air current in your work area.
* A small room or closet that can be closed off is worth the effort to set-up if you will be doing a lot of microbial culturing.
* You can install a UV bulb in a fluorescent light fixture to surface sterilize your work bench if you have an enclosed area. Remember to leave the area when you turn on the UV light source!

Sterile Technique (3)

* All glassware should be cleaned and sterilized before you begin.
* All pipettes, spatulas, and test tube (culture) racks should also be sterilized.
* You can purchase sterile, disposable culture tubes, petri dishes, and pipettes to minimize the quantity of glassware that you have to sterilize.

Sterile Technique (4)

* Don’t forget to wash you hands after you finish cleaning and put on a pair of sterile disposable gloves before you begin.
* Once your work area is clean, your hands are clean, and your glassware is clean and sterile, don’t contaminate the work area by placing “dirty items” such as pencils, pens, notes, or books in the sterile work area.

Media Preparation
Microbiological Media

* The type of growth medium that you use is a function of the organisms that you want to culture. Use a reference book (there are many) to determine the type of medium that is best suited for your organism of interest.
* Common media include Luria Broth (LB), Nutrient Agar, Potato-Dextrose Agar (PDA), Bold’s Basal Medium (BBM)….
Luria Broth

Things to remember:
Assemble all of your chemicals in your work area before you begin.
Accurately weigh each of the dry ingredients in your culture media.
Add each dry culture medium ingredient to the culture flask.
Add distilled (or deionized) water to make the correct volume. Heat AND stir (agar will burn if it is not stirred) until all of the ingredients go into solution. When the media boils, it is ready for sterilization.
Media Sterilization

* There are two reliable methods used to sterilize microbial culture media:
o autoclave
o pressure cooker
* When using an autoclave, use the “wet” setting for sterilizing liquids (flasks, bottles, culture tubes, etc), and use the “dry” setting when sterilizing empty containers, stoppers, etc.

Media Sterilization (2)

* All liquid media should be sterilized for a minimum for 45 minutes at high temperature and pressure. Autoclaves will cycle automatically, but if you use a pressure cooker, set a timer.
* Remember not to tighten the cap or seal on any container; it will explode under high pressure and temperature!
Sterilize for 45 minutes using the wet cycle (autoclave) or at maximum pressure in a pressure cooker. Remember to cover the top of the flask or jar with aluminum foil to prevent contamination when as the media cools.
When using a pressure cooker, don’t over fill the cooker, and remember to weight your containers so they don’t fall over!
Sterilize at high temperature and pressure for 45 minutes before turning off the heat. Remember to allow enough time for the pot to heat up!

Plate Pouring Tips

* Line empty plates along the edge of the work bench.
* Open the petri dish lid at about a 30-45° angle to allow the hot liquid to cover the bottom of the dish. The thermal current created by the hot media prevents bacteria and fungal spores from landing in your clean dish.

Line your sterile petri plates along the edge of the table. Transfer hot media to a small sterile container and pour 15-20 ml of the plate media into each petri plate. The petri plate lid should be open slightly, but not completely open as this increases contamination.
* As the plates are poured, move the filled plates to the back of the table until the plates cool and congeal.
* Once the plates have cooled and the media is firm, store the plates media side-up (bottom) with the lid securely taped or the plates restacked in the manufacturer’s plastic sleeve.
* To increase the shelf-life of the plates, store in a cool, dry environment until they are used (refrigerator).


Inoculating Plates and Culture Tubes

* Clean and surface sterilize your work area as detailed in the section on Sterile Technique.
* Use either disposable inoculation loops or a metal loop that can be heat sterilized to inoculate plates, slants, and liquid culture tubes.
* If using a metal loop, be sure to cool the loop by touching the sterile cooled liquid media or the sterile culture plate before the placing the loop in your live culture. Failure to cool the loop will kill your active microbial cultures!
If gas is unavailable in your lab area, you can modify a standard Bunsen burner to use camp stove propane containers as fuel.

Inoculation of Liquid and Solid (Slant) Culture Tubes

Step 1: Remove the culture tube stopper or cap with one (do not set it down) and flame the mouth of the tube to surface sterilize the mouth. The heated tube surface will generate a thermal current that prevents contamination of the culture.

Step 2: Without setting any of the culture materials on the bench, place the sterile inoculation loop in the culture.

Step 3: Replace cap on the culture tube with the active microbes and put it in the test tube rack.

Step 4: Without setting the loop down, pick-up a sterile fresh culture tube with media with one hand, and remove the cap with the other hand.

Step 5: Flame the mouth of the clean culture tube.

Step 6: Place the inoculation loop containing the microbes in the fresh media and swirl the loop in the loop in the media to ensure even dispersal in the media.

Step 7: If using a solid media slant tube, follow steps 1-5 and then zig-zag the inoculation loop across the slanted surface of the solid media in the tube.
Step 8: Flame the mouth of the newly inoculated culture tube and replace the cap.

Step 9: Place the culture tube in test tube rack.

Step 10: Repeat until all of the sterile tubes have been inoculated. Use a fresh disposable culture loop for each tube or flame the metal loop after each tube has been inoculated.

Step 11: Incubate the culture at the recommended temperature (check with your supplier for growth requirements). If using environmental samples, incubation at room temperature will avoid the accidental culture of human pathogens.

Step 12: Dispose of all culture materials in a biohazard bag and sterilize all old cultures before pouring out cultures and washing culture tubes. Disposable culture dishes should be melted in an autoclave or pressure cooker prior to disposal.

Inoculating Petri Plates

Step 1:Remove the culture tube stopper or cap with one (do not set it down) and flame the mouth of the tube to surface sterilize the mouth. The heated tube surface will generate a thermal current that prevents contamination of the culture.
Step 2: Without setting any of the culture materials on the bench, place the sterile inoculation loop in the culture.
Step 3: Replace cap on the culture tube with the active microbes and put it in the test tube rack.
Step 4: Holding the petri dish lid at an 30-45° angle, work the inoculating loop from the outside of the plate toward the center in a zig-zag pattern that covers approximately 25% of the plate surface (think pie or pizza slice!).
Step 5: Turn the petri plate 90° to the right, dragging the inoculation loop through the last section of the plate, moving from the outside to the inside in a zig-zag motion.
Step 6: Repeat this process twice more until the entire plate surface is covered.

NOTE: If you are trying to isolate individual colonies, each turn of the dish will give you fewer microbes so that you can distinguish individual colonies.

Use of a Plate Counter for Estimating Microbial Populations

Serial Dilution of Environmental Samples or Commercial Cultures

* Serial dilution techniques should be used in the estimation of microbial population sizes.
* Serial dilution involves the use of a known amount (in ml or μl) in a known volume of liquid media.
* A one in ten dilution is made in a new liquid culture tube, and this process is usually repeated several times. The resulting cultures are dilutions of 1/10, 1/100, 1/1000, 1/10,000, for example, of the original sample.
* These cultures are plated on petri plates and incubated at the recommended temperature.

Estimating Microbial Population Size

* After the inoculated plates are incubated for the appropriate time period, the number of colonies per plate are counted.
* Population estimates are obtained by multiplying the dilution factor by the number of colonies per plate. The resulting number is a rate (function) of the initial weight or initial volume used from the environmental sample or culture (per gram soil, per ml or μl of culture).
Counting Plates

* If a commercial plate counter is not available, you can Xerox 1 mm square graph paper and use it as a grid for colony counting. You would need to estimate the total surface area (in mm2) by counting the number of squares in a dish.
* If using a commercial plate counter, touch each colony on the plate with the pen, and the cumulative number of colonies will appear on the display.

Summary

* Different media are used to culture microorganisms, be certain that you are using the appropriate media for your organism.
* Always use sterile technique to prevent contamination.
* Choose the type of media (liquid or plate) appropriate for your investigation or application.
* Sterile liquid culture tubes and media plates can be prepared in advance and stored in the refrigerator for later use (2 weeks for liquid culture tubes, 2 months for media plates).
* Liquid culture tubes, solid slant tubes, and petri plates can be used to culture microbes.
* Media and lab materials should be sterilized prior to use; an autoclave or a pressure cooker can be used in the sterilization process.
* Serial dilution and plate count techniques are used to estimate microbial populations from environmental or commercial cultures.

Microbiological Methods.ppt

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Fundamental Techniques in Microbiology

Fundamental Techniques in Microbiology
Presentation lecture by:Dr Paul D. Brown

Introductory Biochemistry
Fundamental Techniques
* Microscopy
* Staining
* Aseptic technique
* Sterilization and waste disposal
* Media preparation

Microscopy
* Measurement
o Microorganisms are very small
o Use metric system
o Metre (m) : standard unit
o Micrometre = 1 x10-6 m
o Nanometre = 1 x10-9 m
o Angstrom = 1 x10-10 m

Terms Relevant to Microscopy
* Total Magnification
o Eyepiece x objective lens
* Resolution
o Ability of the lens to distinguish two points as separate
o Optimal RP achieved with blue light
o Theoretical limit for light microscope is 0.2 m
* Refractive Index (η)
o Measurement of relative velocity at which light passes through a material.
o η= 1.0 in air
o η (Oil) = η (glass) = up to 1.5
Resolving Power
Transmission electron microscope
Scanning electron microscope
Light microscope
Human eye
R.P. in Angstroms
Resolving Power
Optical Instrument

Types of Microscopes
* Simple: one lens
* Compound: more than one lens

The Compound Microscope
* enters the eye
o sees virtual, inverted image
* further magnif. by ocular
* forms magnified real image
* enters objective
* focuses light on object
* light enters condenser

Objectives

* 10X Scanning Find the object
* 40X High-Dry Focus the object
* 100X Oil immersion Fine focus

The Condenser
* Functions
o Focus light on object plane
o Ensure adequate intensity
* Height of condenser controls
o Uniformity of brightness
o Contrast (minimises “stray light”)
o (Indirectly) angle of light entering objective

Use of Immersion Oil
Condenser Iris Diaphragm
Bright-field Microscope
* Contains two lens systems for magnifying specimens
* Specimens illuminated directly from above or below
* Advantages: convenient, relatively inexpensive, available
* Disadvantages: R.P 0.2 m at best; can recognize cells but not fine details
* Needs contrast. Easiest way to view cells is to fix and stain.

Different magnifications
Special Microscopy Applications
* Dark Field
* Phase Contrast
* Fluorescence
* Electron Microscope
* special condenser diaphragm
o occludes direct light, passes wide angle light
o angle too wide to enter objective
Phase Contrast Microscopy
Fluorescence Microscopy
Electron Microscopy
Stains and Staining
Simple Stains
Differential Stains
* Gram stain
o Crystal violet: primary stain
o Iodine: mordant
o Alcohol or acetone-alcohol: decolourizer
o Safranin: counterstain
o Gram positive: purple
o Gram negative: pink-red

Staphylococcus aureus
Escherichia coli
Gram stain – distinguishes Gram+ from Gram -
* Acid-fast stain
Special Stains
* Capsule stain
* Flagella stain
* Spore stain (Schaeffer-Fulton)

Aseptic Technique
* First requirement for study of microbes
o pure cultures, free of other microbes
* Maintain a clean environment; work close to the flame

Streak plate method of isolation
Sterilization and Waste disposal
Culture media formulation
Types of media

* General purpose
o Allows growth of most bacteria, e.g., Nutrient agar
o Includes organic C, N, vitamins
o May have undefined components e.g., yeast extract, peptone
* Defined
o All components are pure compounds, not mixtures such as yeast extract
o E.g., glucose + (NH4)2SO4 + minerals for E. coli

* Selective
o Favours one organism and limits growth of others
o Lacks some factor(s)
+ E.g., fixed N, to select for N2-fixing bacteria
o Selective toxicity
+ E.g., bile salts to select for Enterobacteriaceae
* Selective via incubation conditions
o E.g., gas composition (e.g., N2, 5% CO2, O2), temperature
* Differential
o Different bacteria/groups give different responses
o E.g., MacConkey agar: has lactose + peptone + indicator (neutral red)
+ lactose fermenters - acid - pink colour
+ non-lactose fermenters use peptone - neutral or alkaline - colourless

Enrichment Techniques

* Increase proportion of desired physiological class
o E.g., N2-fixers; cellulose-decomposers; photosynthetic bacteria
* Culture mixed population in selective medium and/or conditions
o E.g., fixed N-free; cellulose as sole carbon, energy source; anaerobic conditions in light, without organic C
* Sample treatment
o E.g., boil to kill vegetative cells, leaving spores

Fundamental Techniques in Microbiology.ppt

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