Robert Koch is famous for being the first scientist to conclusively identify the etiological agent of any disease (anthrax in this instance). He established a set of criteria which must be satisfied in order to do this, called “Koch’s Postulates.” They are:
1) All diseased animals must display the putative pathogen.
2) The putative pathogen must be isolated in pure culture.
3) The pure culture, when inoculated into a healthy animal, must cause the disease.
4) The putative pathogen must be reisolated from the experimentally diseased animal.
The major challenge in these criteria is to isolate “the putative pathogen” (bacterium) in pure culture. Koch was familiar with the work of Joseph Schroeter, who observed that the surface of a potato slice would develop small colored raised circular spots when left open to the air. Koch reasoned that each of these raised spots must have arisen from a single contaminating cell. As it multiplied, the single cell produced a visible clone of identical cells (a colony) .
He realized that these colonies were pure cultures (clones) of bacteria because each arose from a single cell.
Fortuitously, Bacillus anthracis, the etiological agent of anthrax, was able to grow on potato, and would yield a pure culture by which he was able to satisfy his postulates.
sterile petri dishes, one per two students
clean cutting board
sharp paring knife
tweezers in an EtOH beaker
95% ethanol in a deep beaker
cooked potatoes (not too soft)
1) Pre experiment (understand the experiment and lay it out!):
a) Cross reference in your Notebook the protocol you are following.
b) Describe the specimen with which you are going to inoculate your potato.
c) Label your petri dish in small letters ar the edge of the plate:
i) your initials
ii) the date
iii) the specimen being tested.
2) Sterilize a paring knife by dipping into 95% EtOH, shake off the excess, then briefly flame to burn off the alcohol.
3) Slice a cooked potato about 1/4 inch thick and, using tweezers dipped to sterilize in EtOH and flamed to remove excess EtOH, place the slice on the bottom of a sterile petri dish.
4) Inoculate the surface of the potato with a sample of your choice: wipe a finger across it, lick it, swab the floor with a Q tip, sample your nose with a Q tip, (or pick your nose and wash afterwards…), or leave open to the air for several minutes, etc
Place in 37 C incubator
5) Cover the Petri dish and place in the 37°C incubator in your assigned spot according to your seat number.
6) Incubate for 48 hours, and examine the surface of the potato for any growth.
7) Describe the colonies which appear, and, time permitting, smear and stain to see their morphology. Illustrate what it looks like. Then, if you like, take a picture?
Feta is traditionally made in Greece from ewe’s milk, but I have had success using my goat’s milk. I see no reason why cow’s milk would not work as well. It is a fresh, snow white cheese which is pickled in brine. It is fabulous with kalamata olives and pita bread, as well as in a Greek salad.
By the way, the most popular Turkish cheese called Beyaz Peynir uses essentially the same recipe. We loved it with our breakfasts when we have visited wonderful Turkey. (It may be better not to tell the Turks that it is just like feta, and vice versa… 😉
Thanks to “Lynn” from the Lactobacillus Board for helpful suggestions.
1 gallon fresh goat’s milk (You can use store-bought cow’s milk as well.)
1 Tbl fresh yogurt (I have had most success with Dannon Plain.)
½ tablet rennet, dissolve in 1/4 cup water (I have always used Junket Rennet tablets.)
2 gallon pot with lid (stainless steel with heavy bottom is best, enamel works, but you must stir it!)
1 long bladed knife
2 clean sterile handkerchiefs
Cheese mold: Cut the ends out of a smooth-sided 4 x 5 inch tin can, save one of the cut ends.
Warm 1 gallon of fresh milk in a 1.5 gallon stainless steel pot to 30°C, (86°F)
Here are the results of an experiment at producing home made rennet.
Rennin is an enzyme which, in an acid environment, digests the water soluble milk protein casein into insoluble products. When these precipitate out of solution, the milk coagulates. The test is the famous “clean break” of cheese making.
Here, the abomasum of a suckling kid was cleaned, salted and dried. A small piece (0.75 gm) of it was suspended in warm water (30 C), and added to 1 gallon of inoculated milk. While a clean break was not achieved in three hours, by the evening (about 7 hours) the milk had formed a very firm coagulant.
This is my first attempt at using home made rennet. I am sure that the process and conditions can be improved. Let me know if you have suggestions.
See the bottom of the page for suggestions from Mr. Wolfgang Pachschwöll, of “Hundsbichler company Austria – producer of natural rennet.”
Here are some points of expert advice on making rennet from Wolfgang Pachschwöll of “Hundsbichler company Austria – producer of natural rennet”, sent in response to my initial posting of this page. (Thank you very much Wolfgang!)
1) Do not thoroughly clean out the inside of the abomasum. The “slime” inside contains rennin. Therefore, also no washing nor squeezing.
2) Lightly salt the abomasum, store undried with 30% salt in a closed container to activate the enzyme over three months. (Pepsin, another stomach enzyme, is also secreted in the inactive form (pemsinogen), and activated by acid or enzymatic action.)
3) The traditional way to then dry the abomasum is to inflate it like a balloon and dry by hanging in a cool dark place.
4) Dissolving and activation of rennin occurs best in acid conditions at a cool temperature.
Making paneer (or panir) is a simple exercise in acid/heat precipitation of protein. The only challenge is not to burn the milk while you heat it to hot but not boiling. A thick bottomed stainless steel pot should do, but lacking that, try heating the milk in a water bath so that the volume of water stabilizes the temperature. Here is my recipe for panir:
If you are new to cheese making, please read the page on Beginning Cheese Making for suggestions of easy cheeses to start with.
Swiss cheese is not one of the simpler cheeses to make. The following recipe is still being refined. I believe it is more complex than absolutely necessary, but have not yet performed all the experiments to know how to best streamline it. The eyes were too small and the bite too mild when I made it. If you have experience making Swiss cheese, let us know the lessons your have learned.
One of the major differences between Swiss and other cheeses is that a unique bacterium, Propionibacterium shermanii, is used to ferment the cheese after it is formed into a wheel. This bacterium produces carbon dioxide (hence the bubbles or “eyes” in the cheese), and propionic acid which gives Swiss its unique bite.
Ingredients to turn a gallon of milk into a pound of Swiss cheese:
1 gallon fresh milk
1 tablespoon fresh yogurt (with equal parts L. bulgaricus and S. thermophiles.)
1/4 teaspoon Propionibacterium shermanii culture
1/2 tablet Junket Rennet
Warm milk to 95 F.
Add small amount of milk to the yogurt and P. shermanii cultures, stir to mix, whisk thoroughly into milk, let set 20 minutes.
Meanwhile, dissolve ½ tablet rennet in 1/4 cup fresh cool water
Stir the dissolved rennet into the inoculated milk, cover undisturbed for about 30 minutes until a clean break is achieved. If it takes longer than 30 minutes, use more rennet next time.
Cut the curd by making 1/8th inch vertical cuts in two directions to make long 1/8th inch strips. Then whisk the strips with a pastry whisk so that all levels of the curds are cut. Final curd pieces should be the size of a wheat grain. Maintain temperature at 95 F.
Hold the temp at 95 F for 30-40 more minutes, then slowly increase the temperature with stirring to 125 F. Hold at 125 F for an additional 45 minutes.
Test for completed cooking by squeezing a handful of curds into a ball. If it readily breaks up when rubbed between palms, it is ready.
Let curds settle, dip off some whey.
Dip out the curds into a clean handkerchief suspended in a strainer over a catch bowl.
Pick up the four corners of the handkerchief, dip into whey to loosen curds, then set in cheese hoop.
Press for five minutes, remove, replace cloth, and press for three more hours.
Rinse cloth in saturated salt water, replace in press for three more hours.
Repeat rinsing of cloth in salt water and pressing for three additional hours.
Repeat rinsing of cloth in salt water and press overnight.
Prepare saturated salt water bath: dissolve 5 Tbl salt in 16 oz water (some salt remains undissolved). Pour into a plastic container slightly wider than the cheese, cool the salt solution down to 45 F. Float cheese for two days in this 45 F brine, turning each day, sprinkle salt on surface of cheese. [NOTE: I have recently received an email that suggests this time is too long, that the cheese may become too salty. I am not certain about the finer points of brining the cheese, and am eager to hear any information others may have on the subject.]
Finally, place cheese on board at 50-55 F, 90% humidity. Wipe and dry board daily for 10 days. Wipe the cheese with salt soaked cloth and turn.
Rub the cheese with salt at end of 10 days.
Move cheese to 70 F, 70-80 % humidity.
Wipe with clean salt water 2x per week, continue for a month and a half. Cheese should puff up as characteristic holes form.
See Alcamo, 4th , p 451-465, Campbell’s 5th, pp 524-528, Tortora, Funke& Case, 7th, pp 349-366, 8th: 352-375.
Illustrate the protozoans (first three specimens) at 1000x, and the last, the nematode Trichinella, at 400x (or 100x). As always, scan at lower power to find a field rich in organisms. Take care to maintain correct scale. Label features which can be observed, note diseases they cause.
MASTIGOPHORA: Class of whip-bearing protozoans, in kingdom protista, phylum protozoa
Slide 22 Trypanosoma gambiense (PS 310)
(or view Trypanosoma lewesi PS 300, harder to see)
In an infected blood smear, among the erythrocytes (RBCs), wavy organisms with nucleus, flagella and (hard to see) undulating membrane. A member of the kinetoplastids.
Causes sleeping sickness. Spread by the tsetse fly vector in Central Africa. Forms a chancre at bite, fever, facial edema, lymphadenopathy, CNS symptoms, finally, after several years, fatal coma.
Slide 19 Giardia lamblia (PS 210)
In an infected fecal smear. Bilateral trophozoites: two cells adhered looks like a tear drop with two nuclei appearing like eyes. Note “old man” appearance due to diploid structure. The eyes are the nuclei of two cells.
giardiasis: Most common protozoal disease, 4% of USA population are infected, commonly asymptomatic. Causes small intestine enteritis (nausea, flatulence, eructation, copious, frothy, foul smelling diarrhea). Coats wall of sm intestine, can prevent absorption of nutrients, esp. lipids. Transmitted in feces as cysts, human and wild animals. One cause of traveler’s diarrhea, not killed by Cl2 .
Slide 20 Trichomonas vaginalis (PS 250)
In an infected vaginal smear: Roughly circular, nucleus is barely visible within cell. The flagellum is only occasionally visible. Note squamous cells and polymorphonucleocyts.
8 additional views of T. vaginalis:
Here is a cluster with a polymorphonucleocyte in the field.
Causes vaginitis, when pH is 5.5 or above. Found in GU of males & females. Causes pruritus, copious green/yellow frothy discharge (ingests lactobacilli which raises vaginal pH from 3 to 4.5). 30% of the population is colonized. STD, spread also on towels, toilet facilities, very sensitive to drying.
Plasmodium vivax causes malaria, infecting 250 mil in world, 1 mil die/yr in Africa):
REPRODUCTION: Plasmodium vivax infects salivary glands of Anopheles mosquito: several 100 sporozoites injected with a bite. These migrate to liver where (in 2-4 wks) they are transformed to many thousand asexual spores, merozoites. These infect RBC, form ring stage, asexually generating more merozoites by schizogony (multiple fission), released by lysis (fever). Anopheles mosquito draws blood containing gametocytes (develop from merozoites), sexual reproduction occurs in female mosquito (zygote formed) in salivary glands. meiotic products form sporozoites.
Signs: fever with each coordinated RBC rupture cycle. Last 8 hrs, then 48 hr remission, repeat fever, RBC destroyed, anemia, splenomegaly, “blackwater fever”, RBC fragments block arteries to organs.
PHYLUM NEMATODA (Kingdom Animalia)
At 100x or 400x: Illustrate several cysts in striated muscle (tongue)
Slide 24 Trichinella spiralis (PS 2430)
At 100x or 400x: Illustrate several cysts in striated muscle (tongue) . Note numerous sections through the coiled nematode. The roundworm is suspended in a jelly-like material within the capsule.
Trichinosis is caused by this eukaryote. Encysted larvae occur in pork, bear meat, and flesh of other carnivores. They mate in intestine, lay living larvae. Cross GI track, migrate through blood to muscles (frequently to the eyelids, causing puffiness over the eyes). Can cause heart, kidney failure. Only 150 cases/yr these years in the USA, but >90% of Americans carry Ab, i.e., carry at least a few worms. They are killed by >77 C temperatures, but often survive microwave cooking because of cooler spots within the meat.
A number of interesting controversies have revolved around the differences in milk from various species (especially as to the relative appropriateness for use as milk for humans, notably babies). There are some notable differences in composition of milks according to the USDA’s Handbook of the Nutritional Contents of Foods, by Watt and Merrill, United States Department of Agriculture, Dover Publications, Inc, New York (1973). (See Table I, pp 38 & 39.) Data for sheep, water buffalo and reindeer came from Food Values, Pennington and Church, Perennial Library, (1985)
I have picked out nutrients from their table which have the greatest variation and compiled them in the table below. (100 mL is a little less than 1/3rd cup):
Additional Milk Nutrients from Food Values, Pennington and Church, Perennial Library, 1985
One could write a whole paper on these differences. I think the most interesting are:
Human milk has about 1/3rd as much protein as either cow or goat.
Human milk has twice the carbohydrate as either cow or goat
Human milk has 1/4th the phosphorous as either cow or goat
Human has five times the of vitamin C. This is not surprising, since of these three species, only human are unable to synthesize their own vitamin C, and thus must get it through the diet.
It may be notable that the fat content is about the same for all three species. Also, there are relatively few differences between cow’s and goat’s milk in this table, and yet our experience is that goat’s milk is more easily digested than cow’s. Persons with peptic ulcers have said that it helped considerably to consume goat’s milk.
We do not know, but we know that goat milk has smaller fat globules than cow milk. Besides making the fat content more easily digested, this explains why cream separates much faster from cow than from goat milk.
Also, over many years, we have had parents of young infants come to us for goat’s milk because their infants could not digest either cow’s milk formula, or soy milk formula, and the mother had dried up (or been dried up by the pediatrician!). These parents have been emphatic that their infants thrived on goat’s milk as opposed to cow’s or soy “milk.” The differences in digestibility of goat’s versus cow’s is apparently in some category not listed in this table.
We know that goat’s milk is naturally homogenized, since the cream does not separate nearly as readily as cow’s milk. We have interpreted this as the reason for goat’s milk digestibility. However, we have received email correspondence indicating that human milk does separate overnight, so there is probably more to the story.
The most important consideration in good flavored milk and milk products is the proper handling of the milk from the time it is milked out to the time that it is consumed or made into cheese. It makes me wonder about commercially available goat’s milk, which in my experience has a strong “goaty” flavor. The goat’s milk I produce only has had such flavor problems if the steps given below were not followed. Many people who say they hate the taste of goat’s milk are usually referring to ‘store-bought’ goat’s milk, and find mine mild, sweet and rich. Likewise, many commercial goat cheeses taste like they were cured in the billy pen… NOT to my liking.
Here are the critical factors I have discovered over a couple of decades of keeping goats and making cheese. All of these are aimed at keeping bacterial contamination as low as possible. Undesirable bacteria are what make milk products have an off flavor. The goal in cheese-making is to add beneficial bacteria which produces good flavor while avoiding the rest. Undesirable bacteria abound on goat hair and dander. Removal of these is the goal of careful filtration.
Never try to make cheese out of “turned” or spoiled milk–the unpleasant flavor will linger. Feed it to your pets if they will drink it. Otherwise, put it on the compost pile.
Note that repeated reference is made to complete drying of thoroughly cleansed equipment. The reason is that most bacteria do not survive well on clean dry surfaces exposed to the air.
Avoiding Bacterial Contamination in your Milk Products
Cleanliness/Sterility of Milking Cans and Storage Bottles:
Immediately after milking, rinse equipment in lukewarm water to remove the majority of milk. If you let the equipment sit, the drying milk will glue itself in the cracks and crevices, and will be come a breeding ground for bacteria.
You should carefully wash the rinsed milking cans in very hot soapy water, rinse well, and air dry COMPLETELY. (Do not dry with a towel, it is easy to introduce bacteria this way.) If you have no problems with odor or taste in your milk, actually sterilization of the cans may not be required. But if you are having problems, your implements should be boiled and air dried. I avoid chlorine because of its poisonousness, but in the worst cases, may have to be resorted to.
Essentials of Recommended Cleansing:
Wash implements well in very hot water and soap
Rinse thoroughly in very hot fresh water
Ensure that they are thoroughly air dried before using
If you must use chlorine for sterilization, use as little as possible, and avoid any trace in your milk.
Stages of Milk Handling
I. Setting up Milking Equipment
II. Setting up Goat to be Milked
III. Cleansing Udders
IV. Milking and Feeding
V. Filtering and Recording
VII. Cleansing Equipment after Milking
Keep the milk chilled at 4ºC until ready for use. Do not add warm milk to previously chilled milk. It will encourage any bacteria in the older milk to grow. However, once thoroughly chilled, milk from sequential milkings can be pooled.
Follow these steps and maintaining a temperature of no more than 4ºC in your refrigerator and your milk should keep easily for more than a week without pasteurization. If goat’s milk is kept this long, cream can be skimmed off when making cheese. Freeze this cream immediately after skimming to produce delicious ice cream.
If you don’t follow these steps closely, you risk a number of bacterial contaminations including those of Salmonella, Escherichia coli and reportedly, Listeria.
Cultured buttermilk is probably the easiest and most fool proof fermented milk product to make. (Note that cultured is different than “old fashioned buttermilk.”) All you need is active cultured buttermilk for the starter, and fresh milk for it to act on (store bought is fine). The formation of buttermilk is based on the fermentation by the starter bacteria which turns milk sugar (lactose) into lactic acid. As lactic acid is formed, the pH of the milk drops and it gets tart. Milk proteins, most notably casein, are no longer as soluble under acid conditions and they precipitate out, causing what we recognize as clabbering. Thus the two marked characteristics of buttermilk, its tartness and its thickened nature, are both explained by the presence or the action of lactic acid. Additional by-products of fermentation give subtle variations in buttermilk flavor.
The acidity of buttermilk also explains its long refrigerator shelf life. Acid is a natural preservative because it inhibits the growth of pathogenic bacteria. Thus buttermilk keeps easily for weeks in your refrigerator. If you keep it longer, it may develop mold on the inner walls of the jar. This mold belongs to the same group of fungi which grow on cheese and is not dangerous. Remove it and the buttermilk can still be used for baking. However, because the desired bacteria may have died in older samples, buttermilk older than three to four weeks may not work as an inoculum to make buttermilk.
Ingredients & Equipment
6-8 ounces active cultured buttermilk
Check the label: it needs to say cultured buttermilk, and is not out of date. (The bacteria die down over time)
3 cups whole milk (store bought works. 2% or skimmed too, but less rich.) very clean 1 quart container with secure lid (I prefer Mason jars).
I have used this recipe for years to make buttermilk in large quantities. I like to use it for baking as well as drinking. It makes pancakes, waffles, and cakes rise very well. You can make any volume of buttermilk you like, so long as you hold to the proportions of 1 part buttermilk plus 4 parts whole milk.
Every year for years, I have prepared a gallon of buttermilk (an ingredient in my cornmeal waffles) for an annual waffle breakfast I serve at Clermont College, serving about 120 people. To make a gallon of buttermilk, I add 1 quart buttermilk to 1 gallon of fresh whole milk in a large container, mix, and pour back into the original containers. The next day, the whole five quarts are nicely thickened.
It works because Streptococcus lactis (or a mixed culture of S. lactis plus Leuconostoc citrovorum) ferments the lactose in milk to lactic acid. The acidic pH causes the protein in milk (most prominently casein, pink in the picture below) to precipitate, thickening the liquid. Because much of the lactose has been broken down to lactic acid, buttermilk should cause less of a problem for those who are lactose intolerant.
It may be that buttermilk could be made with a lower proportion of starter (i.e. 1+6 or 1+8. Anyone have experience with this?) However, the 1+4 ratio has worked so well that I have not wanted to mess with the proportions.
Allow a cup of filtered fresh raw milk to sit covered at room temperature until it has clabbered (usually several days).
Place 1/4 cup of the clabbered milk in a pint mason jar, add a cup of fresh milk (does not have to be raw at this point), cover, shake to mix, allow to sit at room temperature until clabbered.
Repeat this transfer of sub-culturing several more times until the milk dependably clabbers in 24 hours. Taste a small amount to confirm that it is tart, thickened, and has no off flavors. It should taste tart not bitter, for instance.
To then make a quart of buttermilk with this culture, add 6 ounces of the buttermilk to a quart jar, fill with fresh milk, cover, shake to mix, allow to sit at room temperature until clabbered.
Sour Cream Recipe
Sour Cream can be made with the same procedure as buttermilk, using one cup of cream mixed thoroughly with 2 Tbl fresh active buttermilk and letting it sit for 12-24 hours at room temperature. The higher butterfat in the cream, the thicker the finished sour cream.
Cultured versus Old Fashioned Buttermilk
“Cultured buttermilk,” commonly available in United States’ supermarkets, is not the same as “old fashioned buttermilk,” about which I get many questions. The latter is the liquid which remains after churned butter is removed. The two buttermilks bear few traits in common. See the following description of churning butter for the differences.
In “olden times,” farm families would let freshly milked milk sit for half a day and skim off the cream which had risen. This cream would be set aside in a cool place, around 50-60 F. Each milking’s cream would be added until several gallons had accumulated.
In the meantime, naturally occurring bacteria in the cream would cause it to slightly sour. This souring increases the efficiency of churning. The accumulated, slightly sour, cream would be churned at the optimum temperature (approximately 58 F) such that the butter was firm enough to separate out, but soft enough to stick together into a mass. The butter was removed, washed in very cold water to remove the remaining milk, and salt worked in to preserve it. The remaining liquid after the butter was removed was called buttermilk. I call it “old fashioned buttermilk,” which is slightly sour, has the consistency of milk, but is slightly paler. It has flakes of butter floating in it.
Commercial manufacturers sometimes add colored “butter flakes” to imitate the old fashioned buttermilk. However, the two products are very different, cultured buttermilk being thick and tart, old fashioned being thin, and slightly acid, depending on how sour the cream got before it was churned.
Microbiology of Buttermilk
See the page on Smearing and Staining of Bacteria to learn how to see these bacteria with a microscope, and the page on Milk Fermenting Bacteria for a demonstration and discussion of Streptococcus lactis, which is the bacterium which performs this fermentation. Below is a photomicrograph of buttermilk which has been smeared and gram stained. Cells of Streptococcus lactis can be seen as purple spots in a row. Casein is the pink mass covering most of the image.