Blot

“ A fair white sheet with handsomely written words will be more welcome to any reader than a blotted, bedaubed page covered with erasures and dirt, even if the matter in each be of equal value and interest. Erasures, blots, interlineations always spoil the beauty of any letter. ”

“ My books were always full of ink blots, always stained and covered with smeared sketches and pictures, which one draws idly when his attention wanders from his task. ”

“ Wash well in distilled water till no more colour is discharged; if necessary, wash carefully with absolute alcohol.
4. Filter a few drops of the stain on to the film, warm as before, and allow the steaming stain to act for one minute.
5. Wash well in distilled water.
6. Dry and mount.
Note.—The flagella of some organisms can be demonstrated better by means of an alkaline stain or an acid stain—a point to be determined for each. Speaking generally, those bacilli which give rise to an acid reaction in the culture medium require an alkali; those which form alkali in cultivation require an acid. ”

“ The blood-platelets may be examined in the fresh state by coating a cover-slip with Deetjen’s agar-solution (boil 5 grms. agar-agar in 500 cc. distilled water, filter hot, and to each 100 cc. of the filtrate add 0.6 grm. sodium chloride, 6-8 cc. of a 10 per cent solution of sodium phosphate and 5 cc. of a 10 per cent solution of sodium diphosphate) . Place drop of blood on this coating and examine on warm stage. For permanent stained preparations bleed into a fixing and staining fluid (equal parts alcohol and ether and Romanowsky’s stain) or use Wright’s stain. ”

“ They are complex and evolved cells. Inside, there are membranous organelles. This cell type can be animal or plant.
Gram’s method or Gram stain: It is a method created by Christian Gram in 1884 to distinguish and differentiate bacteria under the microscope. The first step is the application of a crystal violet stain. Then, there is the decolorization with ether and/or acetone. Gram-positive bacteria retain the first stain, causing it to look violet or dark blue under a microscope. Gram-negative bacteria become decolorized. ”

“ Luxuriant cultures of the desired organism are grown upon inclined agar; the growth is then removed with salt solution and a glass rod, and thoroughly emulsified by shaking in a test-tube, in order to get the germs well distributed and the clumps broken up. The number of germs in a given quantity of the emulsion is then determined by comparing the number of germs and red blood-corpuscles in a mixture of one part emulsion, one part freshly drawn blood, and three parts of normal salt solution. ”

“ The substance within the introduced cell which acts as a stimulus (antigen) to the body cells is called an “agglutinogen.” That “agglutinogen” is present in the cell has been shown by injecting animals experimentally with extracts of cells (bacterial and other cells) and the blood serum of the animal injected showed the presence of agglutinin for the given cell. It will be noticed that the receptors which become the free agglutinins have at least two functions, hence at least two chemical groups. ”

“ The protein used as antigen to stimulate its formation, or some part of the protein molecule (haptophore group) , which acts as stimulus to the cell is spoken of as a “precipitinogen,” both terms after the analogy of “agglutinin” and “agglutinogen.” In fact the specific precipitation and agglutination are strictly analogous phenomena. Precipitins act on proteins in (colloidal) solution and cause them to settle out, agglutinins act on substances within cells which cells are in suspension in a fluid and cause the cells to settle out. ”

“ As previously stated, most of the physiological activities of bacteria are due to the enzymes that they produce. It is evident that for action to occur on substances which do not diffuse into the bacterial cell—starches, cellulose, complex proteins, gelatin—the enzymes must pass out of the bacterium and consequently may be found in the surrounding medium. Substances like sugars, peptones, alcohol, which are readily diffusible, may be acted on by enzymes retained within the cell body. ”

“ They exactly resemble in everything except the size, the globules of the yeast plant, the Torula Cerevesiæ. Now if we examine more circumstantially the analogies of what I would call the Torula Variolæ with the Torula Cerevesiæ, we observe the following corresponding facts.
"What do we accomplish by inoculation as it is called? Simply this. We take on the top of a lancet, or an ivory point, a few of these minute cells or germs, and we put them in their appropriate nidus, the subcuticular tissue, where, after a few days if they find their appropriate nutrient elements, they grow and multiply." ”

“ This clumping is spoken of as “agglutination” and the substances produced in the animal are called “agglutinins.” If blood cells are injected then “hemagglutinins” result: if bacterial cells “bacterial agglutinins” for the particular organism used as “glanders agglutinin” for Pfeifferella mallei, “abortion agglutinin” for Bacterium abortus, “typhoid agglutinin” for Bacterium typhosum, etc.
The phenomenon may be observed either under the microscope or in small test-tubes, that is, either microscopically or macroscopically. ”

“ By diluting the blood serum from the animal to be tested the agglutinins for the related organisms (so-called “coagglutinins” or “partial agglutinins”) will become so much diminished as to show no action, while the agglutinin for the specific organism is still present in an amount sufficient to cause its clumping. Agglutinins are specific for their particular agglutinogens, but since a given blood serum may contain many agglutinins, the serum’s specificity for a given bacterium can be determined only by diluting it until this bacterium alone is agglutinated. ”

“ Just as an unidentified disease in an animal may be determined by testing its serum as above described against known kinds of bacteria, so unknown bacteria isolated from an animal, from water, etc., may be identified by testing them against the blood sera of different animals, each of which has been properly inoculated with a different kind of known bacteria. If the unknown organism is agglutinated by the blood of one of the animals in high dilution, and not by the others, evidently the bacterium is the same as that with which the animal has been inoculated, or immunized, as is usually stated. ”

“ Two other signs of degeneration are the appearance of granules in the body of the cell protoplasm known as metachromatic granules, owing to their different staining propensities, and the polar bodies which are seen in some species of bacteria. Surrounding the mass of mycoprotein, we find in most organisms a capsule or membrane composed, in part at least, of cellulose. This sheath plays a protective part in several ways. During the adult stage of life it protects the mycoprotein, and holds it together. ”

“ In order that it may exert this power it requires the presence of a substance normally present in the serum known as “complement.” The development of these “anti-bodies,” though it has been studied mainly in connexion with bacteria and their toxins, is not confined to their action, but can be demonstrated in regard to many other substances, such as ferments, tissue cells, red corpuscles, &c. In some animals, for example, the blood serum has the power of dissolving the red corpuscles of an animal of different species ”

“ When sterile nutrient gelatine has been liquefied in a test-tube and inoculated with a mixture of bacteria in such a way that the individual micro-organisms are distributed throughout it, and the liquid is poured out on a glass plate and allowed to solidify, the individual bacteria, instead of moving about freely as in a liquid medium, are fixed to one spot, where they develop their own species. ”

“ It will be noted that the quantities of water selected for addition to each set of tubes of nutrient media have been carefully chosen in order to yield workable results even when dealing with widely differing samples. Plates prepared in agar with 0.1 c.c. and in gelatin with 0.02 c.c. can be counted even when large numbers of bacteria are present in the sample; whereas if micro-organisms are relatively few, agar plate 4 and gelatine plate 1 will give the most reliable counts. ”

“ There is great activity in the nucleus, and granules stream out from it towards the lumen of the gland in much the same way, to take a homely illustration, as bubbles in some effervescing drink form at the bottom of the tumbler and rise till the surface is covered with foam. At the right moment these granules are discharged, just as the bubbles on the surface of a liquid break at a slight jog. They are usually not the ferment or enzyme, but its precursor, a substance which only turns into the ferment when it gets outside the cells. ”

“ By this method Gram-staining bacteria are purple; Gram-decolorizing bacteria and nuclei of cells are brown or red.
2. Cells.—These include pus-corpuscles, epithelial cells, and red blood-corpuscles.
(1) Pus-corpuscles are present in every sputum, and at times the sputum may consist of little else. They are the polymorphonuclear leukocytes of the blood, and appear as rounded cells with several nuclei or one very irregular nucleus (Fig. 8 and Plate II, Fig. 2) . They are frequently filled with granules of coal-dust and are often much degenerated. ”

“ If we desire to test any liquid to ascertain whether it contains any proteolytic—i.e., albumen-decomposing—ferments, we place it in a dialysing tube together with albumen, and note whether peptones appear in the liquid surrounding the dialysing tube. If none are present, we may be sure that the tested liquid contains none of the active ferments capable of decomposing albumen. Should we detect the presence of peptones, we may be certain that some decomposition of the albumen has taken place. In our special case the fluid to be tested is blood serum. ”

“ With many bacteria, especially those that affect proteid matter, foul-smelling gases are formed. These are known as putrefactive changes. All organic matter, under the action of various organisms, sooner or later undergoes decay, and in different stages of these processes, acids, alkalies, gases and numerous other products are formed. Many of these changes in organic matter occur only when such material is brought in direct contact with the living bacterial cell. ”

“ By working through some such scheme as the above we are able to detect what quantity and species of organisms, saprophytic or parasitic, a water or similar fluid contains. For, observe what information we have gained. We have learned the form (whether bacillus, micrococcus, or spirillum) , size, consistence, motility, method of grouping, and staining reactions of each micro-organism; the characters of its culture, colour, composition, presence or absence of liquefication or gas formation, its rate of growth, smell, or reaction ”

“ As is well known, blood serum possesses, as a rule, a bactericidal power upon most micro-organisms, and we have every reason to believe in the existence of specific substances in the serum which exert some influence upon the growth and development of micro-organisms, and also upon the toxic products they tend to elaborate. These protective substances—the alexins of Buchner—appear to be proteid in nature, resembling globulins, since they are precipitated from serum by the action of certain strong solutions of alkali salts, as sodium sulphate. ”

“ Transudates are non-inflammatory in origin. They contain only a few cells, and less than 2.5 per cent. of albumin, and do not coagulate spontaneously. The specific gravity is below 1018. Micro-organisms are seldom present.
Exudates are of inflammatory origin. They are richer in cells and albumin, and tend to coagulate upon standing. The specific gravity is above 1018. Bacteria are generally present, and often numerous. The amount of albumin is estimated by Esbach's method, after diluting the fluid. Bacteria are recognized by cultures, animal inoculation, or stained smears. ”

“ With regard to the behavior of their serum and red blood-cells, all human beings fall into one of three groups. In the first group, the red cells are not agglutinable by any other human serum, while the serum is found to agglutinate the red cells of all persons not belonging to this first group. In the second group, the red cells are agglutinated by the serums of the first and of the third groups, while the serum agglutinates the cells of the third group only. ”

“ It is very unusual for sera to agglutinate any other bacteria then the specific one in dilutions as high as 1-80.
Macroscopic Agglutination.—For the macroscopical or sedimentation test, take a series of small tubes (3⁄8 × 3 inches) and deposit 1 cc. of salt solution in each of the series. Now, having taken an empty test-tube, drop 4 drops of serum in [518] it and then add 12 drops of salt solution. This approximately gives 1 cc. of a 1 to 4 dilution of the serum. It is more exact to make the 1 to 4 dilution with a graduated pipette. ”

“ The Bacteria stand sharply apart from the other forms of life, not only, in many cases, by their divergent methods of metabolism, but by morphological characteristics, such as the definite body-form limited by a distinct envelope, the absence of organs for locomotion other than the peculiar flagella, and, above all, by the lack of any differentiation of the body-protoplasm into nucleus and cytoplasm, as in all true cells of either animal or vegetable nature. ”

“ The main use of the microscope in bacteriology is in the study of stained preparations of the organisms. Staining makes bacteria opaque and hence more easily seen than the transparent unstained forms. Some methods of staining also show morphological structures which are either imperfectly recognized in the unstained cell, spores, or are not visible at all—capsules, metachromatic granules, flagella. ”