Bright-Field Light Microscope and Microscopic Measurement of Organisms SAFETY and CONSIDERATIONS

   

Bright-Field Light Microscope and Microscopic Measurement of Organisms SAFETY and CONSIDERATIONS Slides and coverslips are glass. Be careful with them. Do not cut yourself when using them. The coverslips are very thin and easily broken. Dispose of any broken glass in the appropriately labeled container. If your micro scope has an automatic stop, do not use it as the stage micrometer is too thick to allow it to function properly. It may result in a shattered or broken slide or lens

Medical Application

In the clinical laboratory, natural cell size, arrangement and
motility are important characteristics in the identification of
a bacterial pathogen

Materials per Studentcompound microscope
lens paper and lens cleaner
immersion oil
prepared stained slides of several types of bacteria
(rods, cocci, spirilla), fungi, algae, and protozoa
glass slides
coverslips
dropper with bulb
newspaper or cut-out letter e’s
tweezers
ocular micrometer
stage micrometerLearning ObjectivesEach student should be able to
1. Identify all the parts of a compound microscope
2. Know how to correctly use the microscope—
especially the oil immersion lens
3. Learn how to make and examine a wet-mount
preparation
4. Understand how microorganisms can be measured
under the light microscope
5. Calibrate an ocular micrometer
6. Perform some measurements on different
microorganisms

Why Are Prepared Slides

Used in This Exercise?Because this is a microbiology course and most of the microorganisms studied are bacteria, this is an excellent place
to introduce the student to the three basic bacterial shapes:
cocci, rods, and spirilla. By gaining expertise in using the
bright-field light microscope, the student should be able to
observe these three bacterial shapes by the end of the lab
period. In addition, the student will gain an appreciation for
the small size and arrangement of procaryotic cell structure.
One major objective of this exercise is for the student
to understand how microorganisms can be measured under
the light microscope and to actually perform some measurements on different microorganisms. By making measurements on prepared slides of various bacteria, fungi,
algae, and protozoa, the student will gain an appreciation
for the size of different microorganisms discussed throughout both the lecture and laboratory portions of this course

Principles

The bright-field light microscope is an instrument
that magnifies images using two lens systems. Initial
magnification occurs in the objective lens. Most microscopes have at least three objective lenses on a rotating base, and each lens may be rotated into alignment with the eyepiece or ocular lens in which the
final magnification occurs. The objective lenses are
identified as the low-power, high-dry, and oil immersion objectives. Each objective is also designated by
other terms. These terms give either the linear magnification or the focal length. The latter is about equal
to or greater than the working distance between the
specimen when in focus and the tip of the objective
lens. For example, the low-power objective is also
called the 10×, or 16 millimeter (mm), objective; the
high-dry is called the 40×, or 4 mm, objective; and
the oil immersion is called the 90×, 100×, or 1.8 mm
objective. As the magnification increases, the size of
the lens at the tip of the objective becomes progressively smaller and admits less light. This is one of the
reasons that changes in position of the substage condenser and iris diaphragm are required when using
different objectives if the specimens viewed are to be
seen distinctly. The condenser focuses the light on a
small area above the stage, and the iris diaphragm controls the amount of light that enters the condenser.

Immersion lens

When the oil immersion lens is used, immersion oil
fills the space between the objective and the specimen.
Because immersion oil has the same refractive indexas glass, the loss of light is minimized (figure 1.1). Theeyepiece, or ocular, at the top of the tube magnifies
the image formed by the objective lens. As a result, the
total magnification seen by the observer is obtained by
multiplying the magnification of the objective lens by
the magnification of the ocular, or eyepiece. For example, when using the 10× ocular and the 43× objective,
total magnification is 10 × 43 = 430 times.

Procedure for Basic Microscopy:Proper Useof the Microscope

1. Always carry the microscope with two hands. Place
it on the desk with the open part away from you.

2. Clean all of the microscope’s lenses only with
lens paper and lens cleaner if necessary. Do not
use paper towels or Kimwipes; they can scratch
the lenses. Do not remove the oculars or any other
parts from the body of the microscope.

3. Cut a lowercase e from a newspaper or other
printed page. Prepare a wet-mount as illustrated in
figure 1.2. Place the glass slide on the stage of the
microscope and secure it firmly using stage clips.
If your microscope has a mechanical stage device,
place the slide securely in it. Move the slide until
the letter e is over the opening in the stage.

4. With the low-power objective in position, lower
the tube until the tip of the objective is within
5 mm of the slide. Be sure that you lower the tube

while looking at the microscope from the side.

5. Look into the microscope and slowly raise the
tube by turning the coarse adjustment knob
counterclockwise until the object comes into
view. Once the object is in view, use the fine
adjustment knob to focus the desired image.

6. Open and close the diaphragm, and lower and raise
the condenser, noting what effect these actions
have on the appearance of the object being viewed.
Usually the microscope is used with the substage
condenser in its topmost position. The diaphragm
should be open and then closed down until just a
slight increase in contrast is observed (table 1.1).

7. Use the oil immersion lens to examine the stained
bacteria that are provided (figure 1.3a–d). The
directions for using this lens are as follows: First locate

Examples of Bacterial Shapes as Seen with the Bright-field Light Microscope.

(a) Staphylococcus aureus cocci; singular,
coccus (×1,000). 

(b) Bacillus subtilis rods or bacilli; singular, bacillus (×1,000).

 (c) A single, large spirillum; plural, spiralla (Spirillum volutans;×1,000).

 (d) Numerous, small spirilla (Rhodospirillum rubrum; ×1,000).

Table 1.1 Troubleshooting the Bright-Field Light Microscope

Common Problem Possible Correction

No light passing through the ocular Check to ensure that the microscope is completely plugged into a good receptacleCheck to ensure that the power switch to the microscope is turned onMake sure the objective is locked or clicked in placeMake sure the iris diaphragm is openInsufficient light passing through the ocular Raise the condenser as high as possibleOpen the iris diaphragm completelyMake sure the objective is locked or clicked in placeLint, dust, eyelashes interferring with view Clean ocular with lens paper and cleanerParticles seem to move in hazy visual field Air bubbles in immersion oil; add more oil or make certain that oil immersion objective is in the oilMake sure that the high-dry objective is not being used with oilMake sure a temporary coverslip is not being used with oil. Oil causes the coverslip to float since the coverslipsticks to the oil and not the slide, making viewing very hazy or impossible

LIST OF GRAM NEGATIVE BACTERIA

Gram-negative Bacteria

Gram-negative bacteria are bacteria that do not retain the crystal violet dye in the Gram stain protocol. Gram-negative bacteria will thus appear red or pink following a Gram stain procedure due to the effects of the counterstain (for example safranin).

The Gram Stain

In microbiology, the visualization of bacteria at the microscopic level is facilitated by the use of stains, which react with components present in some cells but not others. This technique is used to classify bacteria as either Gram-positive or Gram-negative depending on their colour following a specific staining procedure originally developed by Hans Christian Gram. Gram-positive bacteria appear dark blue or violet due to the crystal violet stain following the Gram stain procedure; Gram-negative bacteria, which cannot retain the crystal violet stain, appear red or pink due to the counterstain (usually safranin).

The reason bacteria are either Gram-positive or Gram-negative is due to the structure of their cell envelope. (The cell envelope is defined as the cell membrane and cell wall plus an outer membrane, if one is present.) Gram-positive bacteria, for example, retain the crystal violet due to the amount of peptidoglycan in the cell wall. It can be said therefore that the Gram-stain procedure separates bacteria into two broad categories based on structural differences in the cell envelope.

Cell envelope of Gram-negative Bacteria

The Gram negative cell envelope contains an additional outer membrane composed by phospholipids and lipopolysaccharides which face the external environment. The highly charged nature of lipopolysaccharides confer an overall negative charge to the Gram negative cell wall. The chemical structure of the outer membrane lipopolysaccharides is often unique to specific bacterial strains (i.e. sub-species) and is responsible for many of the antigenic properties of these strains. Many species of Gram-negative bacteria are pathogenic. This pathogenicity is often associated with the lipopolysaccharide (LPS) layer of the Gram-negative cell envelope.

Mycobacterium

Mycobacterium is a genus of Actinobacteria, given its own family, the Mycobacteriaceae. Mycobacteria have a cell envelope which is not typical of Gram positives or Gram negatives. The mycobacterial cell envelope does not consist of the outer membrane characteristic of Gram negative bacteria, but has a significant peptidoglycan-arabinogalactan-mycolic acid wall structure which provides an external permeability barrier.

Characteristics of Gram-negative Bacteria

Gram-negative bacteria have a characteristic cell envelope structure very different from Gram-positive bacteria. Gram-negative bacteria have a cytoplasmic membrane, a thin peptidoglycan layer, and an outer membrane containing lipopolysaccharide. There is a space between the cytoplasmic membrane and the outer membrane called the periplasmic space or periplasm. The periplasmic space contains the loose network of peptidoglycan chains referred to as the peptidoglycan layer.

 Gram-negative bacteria

• Acinetobacter
• Actinobacillus
• Bordetella
• Brucella
• Campylobacter
• Cyanobacteria
• Enterobacter
• Erwinia
• Escherichia coli
• Franciscella
• Helicobacter
• Hemophilus
• Klebsiella
• Legionella
• Moraxella
• Neisseria
• Pasteurella
• Proteus
• Pseudomonas
• Salmonella
• Serratia
• Shigella
• Treponema
• Vibrio
• Yersinia

The Study Of Microorganisms

The Study Of Microorganisms

As is the case in many sciences, the study of microorganisms can be divided into two generalized and sometimes overlapping categories. Whereas basic microbiology addresses questions regarding the biologyof microorganisms, applied microbiology refers to the use of microorganisms to accomplish specific objectives.

Basic microbiology

The study of the biology of microorganisms requires the use of many different procedures as well as special equipment. The biological characteristics of microorganisms can be summarized under the following categories: morphology, nutrition, physiology, reproductionand growth, metabolism, pathogenesis, antigenicity, and genetic properties.

MORPHOLOGY

Morphology refers to the size, shape, and arrangement of cells. The observation of microbial cells requires not only the use of microscopesbut also the preparation of the cells in a manner appropriate for the particular kind of microscopy. During the first decades of the 20th century, the compound light microscope was the instrument commonly used in microbiology. Light microscopes have a usual magnification factor of 1000 × and a maximum useful magnification of approximately 2000 ×. Specimens can be observed either after they have been stained by one of several techniques to highlight some morphological characteristics or in living, unstained preparations as a “wet mount.”

Light microscopy

Several modifications of light microscopy are available, such as:

• bright field
  

  The specimen is usually stained and observed while illuminated; useful for observation of the gross morphological features of bacteria, fungi, algae, and protozoa.
• dark field
  

  The specimen is suspended in a liquid on a special slide and can be observed in a living condition; useful for determining motility of microorganisms or some special morphological characteristic such as spiral or coiled shapes.
• fluorescence
  

  The specimen is stained with a fluorescent dye and then illuminated; objects that take up the fluorescent dye will “glow.”
• phase contrast
  

  Special condenser lenses allow observation of living cells and differentiation of cellular structures of varying density.

Electron microscopy

The development of the electron microscope and complimentary techniques vastly increased the resolving power beyond that attainable with light microscopy. This increase is possible because the wavelengths of the electron beams are so much shorter than the wavelengths of light. Objects as small as 0.02 nm are resolvable by electron microscopy, compared with 0.25 μm—allowing, for instance, the observation of virions and viral structures. Specimens are observed by either transmission electron microscopy or scanning electron microscopy. In TEM the electron beam passes through the specimen and registers on a screen forming the image; in SEM the electron beam moves back and forth over the surface of microorganisms coated with a thin film of metal and registers a three-dimensional picture on the screen.

Scanning electron micrograph of the spirochete Treponema pallidum attached to testicular cell membranes.ASM/Science Source/Photo Researchers

Advances in microscopes and microscopic techniques continue to be introduced to study cells, molecules, and even atoms. Among these are confocal microscopy, the atomic force microscope, the scanning tunneling microscope, and immunoelectron microscopy. These are particularly significant for studies of microorganisms at the molecular level.

Nutritional and physiological characteristics

Microorganisms as a group exhibit great diversity in their nutritionalrequirements and in the environmental conditions that will support their growth. No other group of living organisms comes close to matching the versatility and diversity of microbes in this respect. Some species will grow in a solution composed only of inorganic salts (one of the salts must be a compound of nitrogen) and a source of carbon dioxide (CO2); these are called autotrophs. Many, but not all, of these microbes are autotrophic via photosynthesis. Organisms requiring any other carbon source are called heterotrophs. These microbes commonly make use of carbohydrates, lipids, and proteins, although many microbes can metabolize other organic compounds such as hydrocarbons. Others, particularly the fungi, are decomposers. Many species of bacteria also require specific additional nutrients such as minerals, amino acids, and vitamins. Various protozoans, fungi, and bacteria are parasites, either exclusively (obligate parasites) or with the ability to live independently (facultative parasites).

If the nutritional requirements of a microorganism are known, a chemically defined medium containing only those chemicals can be prepared. More complex media are also routinely used; these generally consist of peptone (a partially digested protein), meat extract, and sometimes yeast extract. When a solid medium is desired, agar is added to the above ingredients. Agar is a complex polysaccharide extracted from marine algae. It has several properties that make it an ideal solidifying substance for microbiological media, particularly its resistance to microbial degradation.

Microorganisms vary widely in terms of the physical conditions required for growth. For example, some are aerobes (require oxygen), some are anaerobes (grow only in the absence of oxygen), and some are facultative (they grow in either condition). Eukaryotic microbes are generally aerobic. Microorganisms that grow at temperatures below 20 °C (68 °F) are called psychrophiles; those that grow best at 20–40 °C (68–104 °F) are called mesophiles; a third group, the thermophiles, require temperatures above 40 °C. Those organisms which grow under optimally under one or more physical or chemical extremes, such as temperature, pressure, pH, or salinity, are referred to as extremophiles. Bacteria exhibit the widest range of temperature requirements. Whereas bacterial (and fungal) growth is commonly observed in food that has been refrigerated for a long period, some isolated archaea (e.g., Pyrodictium occultum and Pyrococcus woesei) grow at temperatures above 100 °C (212 °F).

Other physical conditions that affect the growth of microorganisms are acidity or basicity (pH), osmotic pressure, and hydrostatic pressure. The optimal pH for most bacteria associated with the human environment is in the neutral range near pH 7, though other species grow under extremely basic or acidic conditions. Most fungi are favoured by a slightly lower pH (5–6); protozoa require a range of pH 6.7–7.7; algae are similar to bacteria in their requirements except for the fact that they are photosynthetic.

REPRODUCATION AND GROWTH

Bacteria reproduce primarily by binary fission, an asexual process whereby a single cell divides into two. Under ideal conditions some bacterial species may divide every 10–15 minutes—a doubling of the population at these time intervals. Eukaryotic microorganisms reproduce by a variety of processes, both asexual and sexual. Some require multiple hosts or carriers (vectors) to complete their life cycles. Viruses, on the other hand, are produced by the host cell that they infect but are not capable of self-reproduction.

The study of the growth and reproduction of microorganisms requires techniques for cultivating them in pure culture in the laboratory. Data collected on the microbial population over a period of time, under controlled laboratory conditions, allow a characteristic growth curve to be constructed for a species
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