Lab Quiz #2 Flashcards
Endospore Stain
The endospore stain is a differential stain used to visualize bacterial endospores.
Because of their tough protein coats made of keratin, spores are highly resistant to normal staining procedures. The primary stain in the endospore stain procedure, malachite green, is driven into the cells with heat. Since malachite green is water-soluble and does not adhere well to the cell, and since the vegetative cells have been disrupted by heat, the malachite green rinses easily from the vegetative cells, allowing them to readily take up the counterstain.
Using older cultures for endospore stains is better because endospores don’t form until starvation, which is a state more likely to be reached by older cultures than newer ones.
Endospore Stain Protocol
- Perform a bacterial smear of Bacillus or the organism you want to stain.
- Place a small piece of bibulous paper over the smear. Saturate the paper with malachite green.
- Heat the slide gently over the Bunsen burner for 5 minutes. Be sure to keep the bibulous paper
saturated with malachite green during heating. If the slide is steaming, you’re okay; if it stops
steaming, add more malachite green! - Remove the bibulous paper from the slide, and rinse the slide gently with water. Dispose of the
used bibulous paper in the trash. DO NOT leave the bibulous paper in the sink or drain! - Counterstain with safranin for 2 minutes.
- Rinse the slide gently with water.
- Carefully blot the slide dry with bibulous paper.
- Observe the slide under the microscope, using proper microscope technique.
Endospores will stain green. Parent cells will stain red.
Endospore Location
The position of the endospore differs among bacterial species and is useful in identification. The main types within the cell are terminal, subterminal, and centrally placed endospores. Terminal endospores are seen at the poles of cells, whereas central endospores are more or less in the middle. Subterminal endospores are those between these two extremes, usually seen far enough towards the poles but close enough to the center so as not to be considered either terminal or central. Lateral endospores are seen occasionally.
Common Endospore Forming Bacteria
The Gram+ rods common in soil include spore-forming bacteria. The genera Bacillus and Clostridium are the best known, and species of these 2 genera are infamous as the causative agents of wound-generated infections (Clostridium tetani, C. perfringens), and respiratory disease (Bacillus anthracis), and food poisoning (C. botulinum, B. cereus, among others). A distinguishing feature of both genera, and a long list of less common others, is production of endospores.
KOH Test
GN results are harder to decipher and trust than are GP because GP species tend to be larger celled, and age of the culture strongly affects outcome with GN microbes. In fact, E. coli results are only trustworthy for the Gram Stain if the cultures are 48 hours old (otherwise, the results are considered Gram dubious).
The KOH test provides a necessary confirmation after a GN result. For a reliable KOH test the organism MUST BE growing on TSA (Tryptic Soy Agar). It uses KOH in an attempt to dissolve the cells.
- Gram+ cells with their tough thick cell walls of peptidoglycan do not lyse.
- Gram- cells with their thinner more porous cell walls lyse. DNA comes out of the cells, and the bacterial smear becomes a viscous, stringy, sticky mess.
KOH Test Procedure
1) Have everything ready for steps 2-4 beforehand, the bacteria should not be allowed to dry on the slide.
2) Aseptically, place one or two large colonies on a slide (use several small colonies or a few loops of cells if needed) from your unknown stock. The bacteria do not need to be from fresh growth, but they should not be so old that many are dead.
3) Add 1 drop of KOH (.3M?) on top of the cells.
4) Let it sit for 1 minute.
5) After waiting for a minute, add your cool loop to the bottom of the mixture and slowly draw the loop up.
6) If you see viscous strings and stuff, the cells are Gram-.
7) If you see a cloudy cellular liquid, the cells are Gram+. Isn’t this simple!
8) Repeat the insertion of the loop and slowly drawing it up to be certain of your results. Caution, using mostly dead or dried cells or inserting a hot loop can distort the test results.
KOH
An inorganic compound and a strong base. It will lyse the cell that’s gram- to extract the DNA, which is why getting some on the skin is dangerous. Gram+ is protected by a thick peptidoglycan wall, which human skin doesn’t have.
Fungal vs. Bacterial Spores
Fungi reproduce by producing cells called spores. While these fungal spores are somewhat resistant to destruction they are not usually pathogenic to humans. Certain bacteria can produce a thick walled spore structure which allows them to survive adverse environmental conditions for prolonged periods of time.
The bacterial spore is more properly called an endospore because its function is to protect the bacterial DNA from destruction by conditions or substances in the environment that destroy non-endospore forming bacteria. Confusion is widespread because many writers use the term spore for both processes while assuming that their readers already know the difference.
Fungal spores are air-dispensed while endospores are not, which is why sporulating fungi is much more likely to present a contamination problem.
Mannitol Salt Agar Selective
The ingredient that makes MSA selective is 7.5% NaCl. This level of salt is too high for the growth of enterics. So MSA is specifically selecting to favor growth of common skin bacteria over contaminants and also against Gram- bacteria like enterics/Gram- rods of the G.I. tract.
Mannitol Salt Agar Differential
It differentiates pathogens from other forms common on the skin. Gram+ cocci of skin are oxygen tolerant forms and have some types that ferment sugars to acid. All species can use glucose, but pathogens are the only ones that can use mannitol. So MSA only provides that one sugar. Pathogenic skin bacteria ferment it and produce acid, while others generate end products that are actually basic. Phenol Red stains neutral and basic cells red, and acidic pathogens yellow.
Hemolysis Types
Gamma - no destruction (Staphylococcus epidermidis)
Alpha - mild destruction often yielding a greenish-brownish cast and weakly defined margin. (Micrococcus Luteus)
Beta - total destruction with clearing and a sharp margin. (Staphyloccoccus Aureus)
Coagulase
It’s a protein enzyme that enables conversion of fibrinogen to form fibrin. It reacts with prothrombin in blood and results in clotting of blood. The fibrin is used to coat the surface of bacteria ipon contact with blood and its proposed that the fibrin helps bacteria resist phagocytosis.
A positive result for the test will result in agglutination (chunks) of plasma. Negative will result in smooth emulsion (suspension of small globules of one liquid in a second liquid with which the first will not mix).
Staphylococcus vs. Streptococcus
Staph is catalase positive (products catalase) while strep is not.
Staphylococcus is a genus of Gram-positive bacteria. Under the microscope, they appear round (cocci), and form in grape-like clusters.
Streptococcus is a genus of spherical Gram-positive bacteria belonging to the phylum Firmicutes and the lactic acid bacteria group. Cellular division occurs along a single axis in these bacteria, and thus they grow in chains or pairs.
Bacteria of the Skin
Skin is a nasty environment for a microbe. It is dry and tough. Hair follicles and glands, are well-defended by secretions (oils, salt). The outer layer is difficult to penetrate, so pathogens capitalize on cuts and punctures. Most skin dwellers tolerate or profit from oxygen, and resist drying. Typically, they have aerobic tendencies and produce oxidase and catalase.
Three common skin bacteria are all Gram + cocci, with cells in bunches or clusters. Staphylococcus is a facultative anaerobe, while Micrococcus is an aerobe. Staphylococcus epidermidis and Micrococcus luteus are harmless; S. epidermidis forms small circular disc-like white colonies, and M. luteus produces lemon-colored ones. Staphylococcus aureus is an opportunist. When induced to become virulent it produces coagulase and clots blood, and is beta-hemolytic. It ferments mannitol to acid (the yellow on MSA plates). It can cause pneumonia, meningitis, chronic bone infections and is best known for boils and hard-to-heal skin infections. It releases a toxin and is famous for food poisoning. About a third of all people carry S. aureus on their skin.
Propionibacterium lives in hair follicles where it feeds on waxy or oily secretions. To break down the food, it secretes acid that inhibits the growth of other bacteria. P. acnes causes the skin infections that lead to pimples and acne. Again, this is a Gram + form, but is pleiomorphic.
Eosin Methylene Blue Media
The media contains Bile Salts which create an environment hostile to the Gram+ bacteria of skin (selective). It also contains 2 dyes, Eosin and Methylene Blue that favor growth of Gram- bacteria and allow for differentiation of lactose fermenters.
Unlike MSA with Phenol Red, EMB media, with its 2 dyes, allows for acid production to free dye molecules such that organisms which ferment lactose take up stain from the Eosin and turn pink while fast fementers of lactose take up dye from Methylene Blue as well and turn a metallic green. Doubly differential!
