Methods of Microbiology

The Metric System (International System of Units)

Do you know the metric system?  You either know metric or you don’t.  It’s like you’re either pregnant or you’re not. Everybody must be familiar with the metric scale.

The basic unit of measurement is the meter which is a unit of measure.  We take a meter and we divide it into 100 (10-2) units called centimeters. Then we have…

millimeter 10-3

micrometer 10-6

nanometer 10-9

angstrom 10-10

Microbiology is measured in centimeters, micrometers and nanometers.  Remember the magnification is the ocular piece lens magnification times the objective lens magnification.  So what’s 200 nanometers in terms of micrometers?  10-6 versus 10-9 which means… the decimal position moves three spaces so… 200nm would be 0.2µm.

What is limit of resolution?  What does that mean?

The resolution is the ability to distinguish detail within an image and is measured in terms of resolving power.  How far apart two points can be and still be distinguished as being separate is the resolving power.  It’s dependent on the objective lens, the wavelength and the refractive index of the material between the lens and specimen.  The shorter the wavelength of the light being used helps, that is why electron microscopes have such high resolving power and can offer more useful magnification.  The refractive index is the reason why we use oil immersion, because the glass becomes nearly invisible when used with oil.

R (Resolving power, sometimes d) = λ (Wavelength) / 2NA (Numerical Aperture)

A better R value is numerically a smaller number.  The shorter the distance is between two points, is associated with a higher R value and ability to see.  The numerical aperture is the light gathering capacity of the objective lens and is dependent on the combination of the lens size and the use of immersion oil and is usually stamped on the side of the lens.  If you know this and the light wavelength, you could calculate the R, the distance between two points that can be distinguished as being separate.

The limit of resolution for a human eye is 200 micrometers.  The limit of resolution for a light microscope is 200nanometers (or 0.2micrometers).

What is the limit of resolution for an electron microscope?  Well it depends on the type of EM.  Scanner EM is about 10-20nm and a Transmission EM (TEM) is about 1-2nm.

You can’t look at a virus with a light microscope because the limit is 200nm and viruses are 20nm.  An EM is needed for this type of scale.  For the record, hospitals don’t have electron microscopes.  They require a full time individual so you won’t find them in hospital labs.  So how do they know if something is affected by a virus?  Tissue samples with viruses can be examined with a light microscope to see changes in cell structure called cytopathogenic effects.  You basically look at cell damage and note what’s wrong.  If it’s infected by a virus, the cell may be rounded rather than elliptical, for example, and can infer the presence of a virus.  If you take it to a EM lab, they could determine which virus it is.

On a TEM, there’s a beam of electrons coming through and forming an image on a plate (ref: page 65 in book).  Electrons have a shorter wavelength than light so they provide greater detail.

With the scanning EM you only see the surface and can’t any internal details beyond.  Specimens for EM require staining with heavy metals to provide contrast and dehydration through a graded series of alcohol.  You have to dehydrate the specimen so that when the beam hits it, it won’t fizzle.  And you can’t just shove it onto a slide or else it’ll explode.  You have to load with metals to stain it afterwards to provide contrast, then cut thin sections with a diamond knife.

Types of Microscopes

Which one is the most widely used?  The Brightfield.  It’s used for gram stains, tissue exams such as pap smears, or the presence of bacteria, ova and parasites in feces.

Phase contrast and darkfield are limited in use.  The darkfield light microscope is for diagnosing syphilis by detecting motile treponemes.  You could see these wonderfully motile spirochetes that have a very accurate way of moving.  It’s difficult to stain syphilis however.

Fluorescence microscopes are used for Direct Fluorescent Antibody (DFA) tests that uses fluorescent dyes called fluorochromes and they emit visible light on exposure to UV for the antibodies.  DFA is not used as widely as the brightfield but still has use.

Atomic force are very detailed and not useful clinically.  You don’t really need to know how wide a cell wall is or look at images of molecules at the atomic level.  It’s more useful for silicon and metals.  Just remember, brightfield is the most useful.

Work up of a clinical specimen

Patient -> Health Care Professional -> Specimen -> Sent to Lab

(~12-24 hrs) Lab -> Gram Stain -> Preliminary Report -> Empiric Therapy ->

(~3-5 days) Lab -> Innoculate to media -> Incubate -> Isolate in pure culture -> Definitive ID -> Sensitivity/Biochemistry -> C & S.

So u have a patient and they go to a health care professional and of course their job is to come up with a diagnosis and treatment.  How are they going to do that?  As you saw with JD LS (page 1 of ch 1) they did a physical examination for vital signs and then they also did what?  He took a sample from a cervical opening and found yellow discharge and that is sent to the lab.  If it’s a bacterium the first thing they are going to do is a gram stain.  The gram stain doesn’t tell you the identity of the organism, it gives you some reasonable assumptions which can then be used by the health care professional to come up with a preliminary diagnosis by way of EMPIRIC THERAPY to know what antibiotic can be used.  The gram stain divides the bacteria into two types based on color which is used to detect antibiotic susceptibilities.  Now this gram stain gives you a preliminary report.  The preliminary report can be available within 12-24 hours.  That could be potentially life saving for someone in septic shock, for example.

After this, inoculate to sterile media, incubate the culture, then isolation in pure culture and you could get definitive identification based on biochemical profile (and also morphology) but more importantly you could say sensitivity.  This would then go back to the health care professional as a C & S report.  The problem with this is that it takes 3-5 days.

That’s all for this section..