NEXT LAB: (7 days later) examine the air-liquid interface in your flask and look for a slimy growth. The slimy growth may only be on the sides of the flask or it may extend across the liquid surface (a pellicle).
Once you think you have an isolate into pure culture, make a bacterial smear slides and Gram stain them (see Protocols for procedures) to allow you to examine the cellular morphology, arrangement, and cell wall structure of these bacteria. Look carefully for clear areas in the vegetative cells indicative of endospores. You will do an endospore stain in a later lab on any isolates that we expect to be spore formers, but look carefully for this preliminary indication of endospores.
Selective and Differential Media for Confirming Gram Stain Results
Selective Media for Gram positive Bacteria
Phenylethyl Alcohol Agar (PEA)
PEA selects for the growth of gram positive organisms by inhibiting the growth of gram negative bacilli. The alcohol in the medium dissolves the gram negative lipid outer membrane that Gram positive bacteria lack. The thin layer of peptidoglycan, characteristic of gram negative bacteria allows entry of the phenylethyl alcohol into a gram negative bacterium, but not into a gram positive bacterium (gram positive bacteria have a much thicker, more impermeable layer of peptidoglycan in their cell wall). Alcohol interfers with DNA synthesis and kills bacteria with a gram negative cell wall structure. This medium is particularly useful at inhibiting the overgrowth of Gram negative Proteus species that tend to swarm (they are highly motile) all over an agar plate and, thus, using PEA medium makes isolating Gram positive organisms easier in a mixed population.
Recipe: 1.80% Bacto Agar, 1.50% Tryptone, 0.50% Phytone, 0.50% Sodium Chloride, 0.25% Phenylethyl Alcohol (PEA).
Postive control organism: Staphylococcus epidermidis
Selective and Differential Medium for Gram negative Bacteria
Eosin–Methylene Blue (EMB) Agar is a selective and differential medium used to enhance selection of gram negative bacteria. The medium contains peptone, lactose, sucrose, dipotassium phosphate, eosin and methylene blue dyes. Colonies produced by lactose non-fermentors are not dark blue or black because the dyes are not precipitated. The growth of gram positive bacteria is generally inhibited on EMB agar because of the toxicity of methlyene blue dye. In the low dye concentration found in this medium, the protective lipid outer membrane of gram negative bacteria prevents entry of this toxic water-soluble dye because the gram negative outer membrane is a phospholipid bilayer that repels many water-soluble substances. The cell wall structure of gram positive bacteria lacks the protective lipid based outer membrane and that makes them more sensitive to the toxicity of methyene blue.
Recipe:1% peptone, 1% Lactose, 0.2% dipotassium phosphate, 0.04% eosin Y, 0.0065% methylene blue 1.5% Agar. final pH 6.9-7.3
Table 2. Colonial appearance on EMB Agar after 18-24 hours at 35°C.
EMB is also a differential medium. Eosin and methylene blue act as indicators to differentiate between gram negative organisms that ferment lactose from those that do not ferment lactose. Most bacteria that ferment lactose form colonies on EMB agar that are dark blue to black due to precipitation of the dyes by the acid by-products of lactose fermentation. Lactose fermentors have dark colonies from dye precipitation while non-fermentors have light colored colonies. Additionally, E. coli often gives a characteristic green-metallic sheen on EMB, making this medium somewhat differential for E. coli to help distinguish it from other lactose fermenting gram negative bacteria.
Recipe: 0.04% Eosin Y, 0.0065% methylene blue, 1.0% peptone, 2.0% lactose, 0.2% K2HPO4, 1.5% agar, pH 7.1
||purple with black center/ green metallic sheen
||dark centered colonies/ sometimes a metallic sheen
||pink colonies/ no metallic sheen
Reference: Dehydrated Culture Media and Reagents for Microbiology. DIFCO Laboratories, Detroit, MI. 1984.
Differential Medium For Assessment of Soil Exoenzymes: Amylase, Cellulase, Phosphatase
Nutrient Agar (NA) General Purpose Medium is used to determine comparative number of total culturable bacteria: and for growth of non fastidious organisms once in pure culture. For plate counts use your P200 micropipet and sterile tips, dispense 100µl of a soil extract dilution (choose a dilution that should give you between 30-300 CFUs) onto a pre-labeled Nutrient agar plate. Use a sterile, disposable spreader to evenly distribute the diluted soil extract all over the culture plate. Repeat for two other dilutions (one 10fold more and one l0 fold less dilute).
Nutrient Agar General Purpose Medium:
0.3% Beef extract, 0.5% Peptone, 1.5% Agar at pH 6.6- 7.0 at 25°C.
Starch Medium is used to determine the % of amylase producing (starch digesting) culturable microbes when compared to the total number counted on NA: Using your P200 micropipet and sterile tips, dispense 100µl of a soil extract dilution (choose a dilution that should give you between 30-300 CFUs) into the center of a pre-labeled Nutrient agar plate. Use a sterile, disposable spreader to evenly distribute the diluted soil extract all over the culture plate. Repeat for two other dilutions (one 10 fold more and one l0 fold less dilute).
Starch medium :
2.5% (wt/vol) soluble starch in Nutrient Agar
Reference: Beishir, Lois. 1996. Microbiology in Practice 6th ed. HarperCollins Publishers Inc. New York. Module 33: 301-306.
Cellulose Medium is used to determine the % of cellulolytic microbes (those producing cellulase) when compared to the total number counted in NA : Using your P200 micropipet and sterile tips, dispense 100µl of a soil extract dilution (choose a dilution that should give you between 30-300 CFUs) onto a pre-labeled plate of Cellulose medium. Use a sterile, disposable spreader to evenly distribute the diluted soil extract all over the culture plate.
Cellulose Congo Red Agar:
0.05% K2HPO4; 0.025% MgSo4; 0.188% ashed, acid washed cellulose powder; 0.02% Congo red, 0.5% Noble Agar, 0.2% gelatin, 10%(vol/vol) sterile soil extract (Soil extract prepared as follows:105 g of air-dried sieved soil and 660 ml of deionized water are placed in a 1 litre bottle and autoclaved once at 15 psi for 15 minutes, then again after 24 hours. The contents of the bottle are left to settle for at least a week and then the supernatant is decanted and filtered. The final pH should be 7.0 - 8.0.)
Reference: Hendricks, Charles W., Doyle, J.D., Hugley, B. (1995) A New Solid Medium for Enumerating Cellulose-Utilizing Bacteria in Soil. Applied and Environmental Microbiology. May: 2016-2010.
Phosphate Medium (Pidovskaya medium) is used to determine the % phosphate solubilizing microbes (those producing phosphatases) in a soil community: Using your P200 micropipet and sterile tips, dispense 100µl of a soil extract dilution (choose a dilution that should give you between 30-300 CFUs) onto a labeled Pidovskaya medium plate.
1.0% glucose, 0.05% yeast extract, 0.01% Calcium Chloride (CaCl2), 0.025% Magnesium Sulfate (MgSO4.7H20), 0.251% Calcium Phosphate [Ca(PO4)], 2.0% agar.
References: Pikovskaya, R.I. 1948. Mobilization of phosphorus in soil in connection with the vital activity of some microbial species. Mikrobiologiya 17, 362-370, modified by Pranjal Baruah (2007) Isolation of phosphate solubilizing bacteria from soil and its activity. Biotechindia.files.wordpress.com/2007/12/isolation.pdf.
Incubate all cultures at room temp until mature colonies have formed and then refrigerate before the bacteria overgrow.
Count the total number of colonies on the Nutrient Agar plate that has between 30-300 colonies and record the dilution. Assess total culturable CFUs for that dilution.
Find the starch plate with between 30-300 total colonies and note the dilution. Flood the starch plate with a thin layer of iodine and count the number of colonies that show starch digestion activity as a clear zone or non-blue halo around the colony. Iodine turns from reddish brown to blue-black in the presence of starch; therefore, bacteria in those colonies that do NOT turn black have produced and secreted amalyase enzyme that digested the starch in the area around the colony into simpler sugar. Those bacteria that do not produce amalyase are unable to break down starch, revealed by the color change to blue-black in the medium surrounding those colonies. Record the number of starch digesting organisms and the dilution.
Find the cellulose plate showing between 30-300 total colonies. Count the number of colonies that show cellulose digestion activity by looking for positive digestion as a clear zone or halo around the colony. Record as number of cellulose digesting organisms in that dilution.
Do the same for the assessment of number of phosphate digestion microbes in a particular dilution. The positive colonies will be red that show phosphate solubilizing activity.
Calculating the % of digestion positive microbes in the total culturable population
Use the soil extract dilution on the plates counted to normalize all the calculations to CFUs/gram of soil (wet weight) for each assessment medium. If you divide the number of colonies counted by the volume of inoculum plated, times the dilution factor of that inoculum, you will obtain the number of that type of bacteria per gram of soil.
For example, if you counted 150 colonies on the 10-3 plate the calculation is:
150/(0.1ml vol. of inoculum*1X10-3dilution)= 150X104 which in scientific notation is written as 1.5X106 CFU/gram
Once you calculate the total number of aerobically culturable bacteria (cfu/g) on the general purpose media, you can determine the % of the total number able to solubilize phosphate by dividing the number of phosphatase positive colonies by the total number of culturable colonies---if the colonies counted are compared from the same plate dilutions.
This calculation of the % of cultured bacteria that are positive for each tested enzymatic activity: (# positive colonies/total count on nutrient agar X 100) gives you a sense of the prevalence and variety of soil organisms in a community with particular substrate utilizing potential.
Medium For Assessment of motility and ability to reduce nitrate
Motility and nitrate reduction medium
Mannitol Nitrate Motility Medium
1% Casein Peptone, 0.75% Mannitol, 0.1% Potassium Nitrate, 0.004% Phenol Red, 0.35% Bacteriological Agar. pH 7.6 at 25°C
Mannitol Nitrate Motility Medium is a semisolid general purpose medium that provides nitrogen, minerals, amino acids and nutrients from the Casein peptone. The fermentable carbohydrate, Mannitol, provides an energy source. Potassium Nitrate provides additional nutrients and organisms capable of reducing nitrate show increased motility. Phenol red is a pH indicator. Agar is included as a solidifying agent.
The inoculating needle is used to introduce a single deep inoculum to the center of the soft agar pour with stab technique. Motile bacteria diffuse away from the line of inoculation clouding the otherwise transparent medium. Non motile organism only grow along the stab line. If mannitol is fermented the medium changes color from red to yellow. After incubation, Gries reagent (2 drops of solution A and 3 drops of solution B, are added to the surface of the medium to test for Nitrate reduction. Nitrate-negative organisms are unable to reduce nitrates and no color change is observed after adding the reagent. If the organism is nitrate positive a pink or red color indicate that nitrates have been reduced to nitrites.
Gries Reagent: Solution A: 0.8% Sulfanilic Acid in 5N Acetic Acid. Solution B: 0.001% Alpha-Naphthylamine in 5N Acetic Acid.
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