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I was wondering if potato dextrose agar was a suitable environment for bacteria. Does it have all the requirements bacteria need or is it limited? Specifically: Escherichia coli, Staphylococcus aureus, Enterococcus faecium, Pseudomonas aeruginosa.
Without knowing specifics about the bacteria I question this will be kept vague.
Generally speaking though, yes, some bacterias can be cultured using a PDA plate. The plate contains potato starch agar and dextrose. Sometimes Tartaric acid is added to stop unwanted bacterial colonies from forming, which again highlights how possible it is for them to form on the plate. The mechanism of action of the Tartaric acid is to lower the pH, out of the physiological range of most bacteria.
Sabouraud Dextrose Agar (SDA) – Composition, Principle, Uses, Preparation and Colony Morphology
Sabouraud Dextrose Agar (SDA) is used for the isolation, cultivation, and maintenance of non-pathogenic and pathogenic species of fungi and yeasts. SDA was formulated by Sabouraud in 1892 for culturing dermatophytes. The pH is adjusted to approximately 5.6 in order to enhance the growth of fungi, especially dermatophytes, and to slightly inhibit bacterial growth in clinical specimens.
Potato Dextrose Agar (PDA) – Preparation, Principle, Composition and Uses
It is a special type of agar used to cultivate fungi. It is a general purpose medium used to cultivate yeast and mold. To inhibit growth a certain type of acid or antibiotic is used. It is used for different purposes such as:
- Used to detect the presence of mold and yeast in the prepared foods and dairy products.
- Used to cultivate yeast and mold from a clinical specimen.
- PDA, when mixed with tartaric acid, is useful for microbial examination of dairy and food products.
- PDA, when used with chloramphenicol is used for selective cultivation of fungi from a mixed sample.
- PDA, when mixed with chlortetracycline is helpful in the microbial enumeration of mold and yeast from cosmetic products.
- Potato Dextrose Agar with Chlortetracycline is recommended for the microbial enumeration of yeast and mold from cosmetics.
- It aids in the cultivation and differentiation of pathogenic and non-pathogenic fungi. (1, 2, 3, 4, and 5)
Image 1: The image on the left shows A. flavus growth on potato dextrose agar while the one on the right is P.chrysogenum.
Image 2: The photo above is the standard potato dextrose agar plate.
What is the principle of potato dextrose agar?
It consists of dextrose and dehydrated potato infusion, which encourage luxuriant fungal growth. The solidifying agent is agar. To inhibit the growth of bacteria, the pH level of the medium is lowered using a specific amount of 10% sterile tartaric acid.
To inhibit the overgrowth of competing microorganisms from the mixed specimen, a selective agent is used in the form of chloramphenicol. It inhibits overgrowth and at the same time permit the isolation of fungi. The acidified medium should not be reheated as doing so will hydrolyze the agar making the agar fail to solidify. (5, 6)
What are the components of potato dextrose agar?
- Potato extract
- Supplement can be added in the form of tartaric acid, chlortetracycline, and chloramphenicol. (5, 6, and 7)
How do you make potato dextrose agar media?
- Potato infusion – an unpeeled and sliced potatoes should bring to boil in a liter of distilled water for about 30 minutes.
- Using a cheesecloth, filter the potato infusion and save the effluent.
- Mix the infusion with agar, dextrose, and water and bring to boil to dissolve.
- Autoclave at a temperature of 121 degrees Celsius for 15 minutes.
- Dispense about 25 ml into the sterile petri dish.
- The final pH is 5.6 ± 0.2.
- If you are going to use a commercial medium powder, you should add 39 grams of potato dextrose agar powder to a liter of distilled water. Bring to boil and mix continuously to dissolve the powder. Autoclave at the same temperature and timeframe.
- If supplements are added, they should be in a controlled amount – chlortetracycline is 40 mg, chloramphenicol is 25 mg, and tartaric acid is 1.4 mg.
- To process the specimen, stream the specimen being studied on the medium using a sterile inoculating loop.
- The plate should be incubated at 25 to 30 degree Celsius in an inverted position.
- The culture should be examined every week for fungal growth. The culture should be kept for at least six weeks before a negative result is made. (1, 5, 9, and 10)
Image 3:An Aspergillus growth on potato dextrose agar.
Characteristics of colony on potato dextrose agar.
- Yellow-green spores that appear to be powdery are visible on the upper surface while the lower surface is reddish-gold. – Aspergillus flavus.
- The upper surface appears olive-green with a sterile white margin. The lower surface appears wrinkled with a distinct orange to red color. – Penicillium chrysogenum.
- The colony appears thick and velvety. It appears cream white on the surface and radially furrowed on the lower surface. – A candidus.
- The colony appears velvety with white and black spores on the surface. The lower surface is yellow and heavily furrowed. – A niger.
- The colony is dirty white on the surface with yellowish pores at the center. It looks velvety and the lower surface has a distinct orange to a chocolate color. – A sulphureus.
- It has a floccose texture with a surface color of white to orange-cream with green. The lower surface looks bright orange and heavily wrinkled. – A versicolor.
- The surface color is dark green with a velvety texture. The lower surface is colorless to cream in color with a shallow center and a furrowed raised margin. – Penicillium corylophilum.
- It has a velvety texture with a dark green surface. The lower surface appears yellow and radially furrowed. – P. expansum.
- The colony has a floccose texture and appears pink on the surface. The lower surface is magenta-red to violet. – Fusarium oxysporum. (2, 4, 7, and 10)
Is potato dextrose agar selective or differential?
A potato dextrose agar is both a selective medium. However, there are a few limitations. For complete identification, a biochemical, molecular, mass spectrometry, and immunological testing should be done on colonies from pure culture first.
Can i grow bacteria on potato dextrose agar? - Biology
By: Jasalavich, C.A., and G.L. Schumann. 2001. Who Done It? Or what's that brown fuzzy stuff on my plum? The Plant Health Instructor.
Modified by: Rachel Hughes & Kirstin Bittel
• Protocol Sheet
• stone fruit (peaches, nectarines, plums, cherries)
• potato dextrose agar plates (3 options):
1) purchase pre-made plates (no additional materials needed)
2) make plates from purchased dehydrated potato dextrose agar medium
3) make plates from potatoes, dextrose and agar
• For options (2) and (3) flasks, distilled water, autoclave, and Petri plates will be needed.
• dissecting needles
• scalpels or single-edged razor blades
• 95% alcohol
• alcohol lamp or candle flame to sterilize dissecting needles and blades
• 10% (v/v) commercial bleach solution
• sterile distilled water
• paper towels
• plastic box with lid
• plastic bags with twist ties
• dissecting microscope
• compound microscope
• microscope slides
• cover slips
• dropper bottle of distilled water
Via laboratory experience, students use Koch’s postulates to determine the cause of disease in stone fruits. Students explain both the root cause of disease in fruits as well as how the cause was discovered.
Students will be able to:
i. Use Koch’s Postulates to determine that a specific organism is the root cause of a specific disease and identify what Koch’s Postulates are within a protocol.
ii. Describe symptoms and signs of diseased fruit. Isolate fungal pathogen onto a nutrient medium.
National Science Education Standard:
Content Standard G
• Science as a Human Endeavor
• Nature of Scientific Knowledge
Both plants and animals can become ill due to pathogens. These disease causing pathogens can be living organisms (bacteria, viruses, or fungi) as well as abiotic agents (for example air pollution). Robert Koch (1843-1910) devised a scientific method to confirm causation of disease by a microbe. His criteria are referred to as Koch’s postulates. Although still commonly used, molecular methods have added a new dimension to disease identification. Some agents of diseases that could not be identified by Koch’s postulates can now be identified through molecular methods.
Monilinia fructicola, which causes brown rot of stone fruit (fruits with pits i.e. plums, peaches, etc), is an easily available fungal pathogen that can be used for a simple demonstration of the Germ Theory of Disease without the need for culture plates or several weeks of class time. A discussion of Koch’s Postulates and their implications can be included. Infected fruit can be obtained at supermarkets or farmers’ markets, or freshly infected fruit can be produced as described above.
Students can use spores from the infected fruit to inoculate healthy fruit. They should also prepare disinfested, wounded fruit as controls. Both fruit should be incubated in separate plastic bags for five to seven days at room temperature. When available, cherries can be used to provide numerous fruit for less cost than a similar number of plums or other stone fruit. Take care to select sound fruit for the experiment. Slightly under-ripe fruit are more likely to be disease-free.
Related and Resource Websites
Quiescent infections caused by Monilinia fructicola shown as small flecks on prune fruit.
(Original image belongs to Ogawa and English, 1991).
Preparing Potato dextrose agar plates
1. Potato dextrose agar plates or potato dextrose dehydrated medium can be purchased from Carolina Biological Supply Co. (http://www.carolina.com) and Ward’s Natural Science Establishment, Inc. (http://www.wardsci.com). If the dehydrated medium is purchased, directions for preparation of plates will be included.
2. Potato dextrose agar plates can be prepared from potatoes, dextrose, and agar according to the following directions:
3. Boil 200 grams of peeled and sliced potatoes in 1 liter of water until the potatoes are soft. Strain through cheesecloth and adjust the filtrate to 1 liter with more distilled water.
4. Add 10 to 20 grams dextrose and 12 to 17 grams agar. Autoclave 15 min at 121º C.
5. Pour autoclaved medium into sterile Petri plates. Makes approximately 40 plates.
Preparing 10% (v/v) bleach solution
Preparing fruit with brown rot for classroom use
1. For teachers who do not want to maintain or purchase cultures, it’s easy to find this fungus just by buying stone fruit (peaches, nectarines, plums, cherries) and leaving them at room temperature in a plastic or paper bag. They are often already infected, and the infection will develop within a week, resulting in obvious brownish spores on the fruit surface. Isolations from these fruits may be contaminated with bacteria and other fungi, so a more successful lab for students can be accomplished by using fruits that have been deliberately inoculated.
2. Prepare fruits about 1 week before they are needed. Disinfest (surface-sterilize) firm, healthy stone fruit for 30 min. in 10% (v/v) bleach solution. Rinse with sterile, distilled water.
3. Using a sterile dissecting needle, scrape spores from a culture of Monilinia fructicola or a fruit with brown rot and stab each fruit four to six times.
4. Incubate at room temperature in a moist chamber (plastic box lined with paper towels moistened with sterile, distilled water) with the lid not tightly closed. Check daily for fungal development, which will vary with temperature in the lab and the ripeness/susceptibility of fruit. Refrigerate the box of infected stone fruit if necessary to preserve good disease development for student use (i.e. don’t let the brown rot completely destroy the fruit).
5. Although the moist chamber as described above does not start out as a completely sterile environment, because you do not sterilize the plastic box or the paper towels, it does provide an environment adequate to favor the growth of the pathogen over other organisms. A clean plastic box and fresh paper towels usually do not introduce problems.
6. Infected fruit can be allowed to dry at room temperature to form a “mummy.” It will probably be possible to use scrapings from the mummy to begin the disease again when needed for another class.
Immediately before these lessons:
1. As students enter the room, have the following question on the board for students to respond to: “Can plants get sick? Why or why not?”
2. Allow students a few minutes to record and share their thoughts with the rest of the class.
3. In their laboratory groups have students observe the diseased fruits you prepared earlier. Ask them to describe the symptoms and signs of the diseased plums (or other stone fruit). Have them examine the suspected pathogen carefully both macroscopically and microscopically and make notes about and drawings of what they see. They will want to refer back to these recorded observations in later steps.
4. Ask students to consider how the diseased plum looks compared to a healthy plum. Is something growing on the plum? Are some parts of the plum softer or firmer? What is the color of the mycelium (hair-like, non-reproductive fungal growth) and spores? Microscopically, what are the characteristics of the mycelium? Is it septate, i.e. does it have internal cross walls that divide the hyphae into compartments? Or does the mycelium just look like long tubes without any internal cross walls? Do you see any spores? What are their shape, color, and size? Would you recognize this fungus if you saw it again? (See below.)
5. Ask students how they would test to ensure that indeed it was the fungus that was causing the disease. What variables and controls might they need to consider? Have students write these tentative ideas down. Then ask students to talk within their groups about what they think might be important factors to consider. As a class, discuss some of the ideas that students think would be central to testing.
6. Explain to the students that Robert Koch (1843-1910) devised a scientific approach to confirm causation of disease by a microbe. His criteria are referred to as Koch’s postulates. [The students probably alluded to some of Koch’s postulates in their ideas about how to eliminate the cause of the fungus as the root of the disease.]
7. Share with students Koch's Postulates:
I. The diseased host is observed for signs of the causal organism and symptoms of the disease the causal organism is shown to be associated with all diseased individuals.
II. The causal organism is isolated into pure culture and described.
III. This pure culture of the suspected pathogen is inoculated into a healthy host and shown to cause the same disease symptoms and signs as originally observed in Step 1.
IV. The same causal organism is re-isolated into pure culture from the inoculated diseased host and shown to be identical to the organism described in Step 2.
8. Explain to the students that they are going to use Koch’s postulates to determine the cause of the disease in the stone fruit. (Step 4 can be eliminated if time is limited). However, Koch’s postulates are not clearly identified in the protocol. As they carry out the protocol they should identify which one of Koch’s postulates each step refers to.
1. To isolate the probable pathogen on a nutrient medium, e.g. potato dextrose agar (PDA):
a) Cut four small (2 mm x 2 mm) pieces of infected fruit tissue.
b) Disinfest briefly by immersing the four pieces of tissue for 15, 30, 45, or 60 sec in 10% (v/v) bleach solution.
Note: This is done to remove any surface contaminants without killing the pathogen deeper in the tissue. Since it is not known exactly how long this takes, several different times are chosen to ensure a successful isolation of the pathogen. You want to disinfest the tissue of any contaminating organisms, but not kill the fungal pathogen.
c) Sterilize forceps by briefly passing them through a flame and allow them to cool. Using sterile forceps, remove the tissue from the bleach and blot dry on a paper towel.
d) Place each piece on the surface of the PDA agar in the Petri plate. Minimize the time that the medium in the plate is exposed to possible contamination from spores in the air.
e) Incubate at room temperature for five to seven days.
2. Describe the isolated pathogen in culture both macroscopically and microscopically. Record these observations as words and drawings. Do you think this is the same organism that you observed on the diseased fruit in Step 1?
3. Use the isolated pathogen to inoculate healthy plums as follows:
a) Immerse 2 healthy plums in a 10% (v/v) bleach solution for about two minutes. This disinfests the fruit of any surface contaminants. In the original fungal isolation onto PDA, small pieces of cut fruit were placed in the bleach solution for a shorter time to avoid killing the pathogen deeper in the tissue. The 2 minute time for whole fruits can be used because the intact skin of the fruit protects the inner flesh from the chlorine. Remove and dry with paper towels. Make a V-shaped cut with a sterile blade on the surface of the first plum. Place loosely in a plastic bag with a moistened paper towel, close with a twist-tie and label. This is the control plum.
b) Repeat with the second plum with this change: inoculate the wound with spores from your isolate using a sterile dissecting needle.
c) Incubate for one week and record your observations of any symptoms and signs that develop on each fruit.
4. Koch's postulates require that the pathogen be isolated from the inoculated fruit-as in Step 9 (step 1 on the student protocol sheet) to determine if it is the same organism that was originally observed on the first diseased fruit.
5. Once students have had time to allow the disease to grow on the healthy fruit and confirm the cause of the disease, bring the class together to discuss the results and to identify how they thought Koch’s postulates were addressed in the protocol.
6. Students may wish to design experiments to further investigate factors that affect disease. For example:
1) What is the effect of temperature on infection and disease development?
Inoculated fruit can be placed at room temperature and in a refrigerator for a simple comparison. Why do we refrigerate most fruits and vegetables after purchase?
2) What is the effect of wounding on infection and disease development?
Spores can be applied to wounded and non-wounded fruit for a simple comparison. What are some potential sources of wounds in commercial fruit production, harvest and shipping?
3) What is the host range of Monilinia fructicola?
Students can bring in healthy fruits and vegetables for inoculation to determine which ones are susceptible to brown rot. Stone fruit are the common hosts of this fungus, but ripe apples and pears sometimes develop the disease. Which species develop brown rot when inoculated with Monilinia fructicola and which ones do not develop the disease?
- Are students able to identify variables and controls that they should consider when attempting to identify the cause of disease?
- Can students identify which steps in the protocol are associated with which of Koch’s Postulates?
- Are students able to isolate the pathogen and if not, are they able to identify what might have gone amiss in their procedures?
In their science notebooks, have students write a reflective conclusion. What did they learn? What new questions do they have? How does the lab connect to “real life?”
The Community Outreach and Education Program is part of the Southwest Environmental Health Sciences Center: an NIEHS Award
Sabouraud Dextrose Agar (SDA) Preparation
Sabouraud dextrose agar is a mycological culture media which is used for the selective cultivation and isolation of fungi from both clinical and non-clinical samples. Sabouraud dextrose agar (SDA) is unique and different from most bacteriological culture media (used for cultivation and isolation of bacteria) because SDA is usually supplemented with antimicrobial agents particularly antibiotics such as chloramphenicol – which inhibits the growth of bacteria. Cyclohexamide is also included as a supplement in SDA during its preparation and cycloheximide helps to inhibit the growth of saprophytic fungi while allowing only the pathogenic fungi being sought for in the sample to grow. Sabouraud dextrose agar is generally recommended for cultivation of fungi in the microbiology laboratory. Another mycological media that performs similar function as SDA is potato dextrose agar PDA), which can also be used in place of SDA.
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In principle or in practice you will see: SDA+Chloramphenicol SDA+ Gentamicin SDA+Chloramphenicol+Cyclohexamide SDA+Chloramphenicol+Tetracycline et cetera. These different names of SDA show the different formulation of SDA, and they also indicate the types of antimicrobial agents that was incorporated as a supplement during its preparation by the researcher. So depending on your choice of antibiotics to use, the name of your SDA after preparation is up to you to define. In this section, the preparation of Sabouraud dextrose agar will be elaborated.This is how Sabouraud dextrose agar looks like after preparation
Components of Sabouraud dextrose agar
The components of Sabouraud dextrose agar base required for Sabouraud dextrose agar preparation include:
- Agar – which is the solidifying agent
- Digest of soyabean meal
- Dextrose / glucose – which is the source of energy and carbon
- Peptone – which is the source of nitrogen and vitamin
- pH – which is usually adjusted to 7.0 at 25 °C (or 77 °F).
Additional requirements: These include antibiotics such as:
You require these materials to prepare your Sabouraud dextrose agar:
- Sabouraud dextrose agarpowder (usually comes in 500 g),
- Vial of chloramphenicol (5 mg),
- Vial of cyclohexamide (5 mg),
- Autoclave, conical flask, measuring cylinder, beaker, stirring rod,
- Bunsen burner, incubator, refrigerator, wire gauze, spatula,
- Weighing balance, timer, cotton wool, aluminium foil, distilled water, Petri dish
STEP BY STEP PROTOCOL TO PREPARE SABOURAUD DEXTROSE AGAR
Penicillium Antibiotic Effect
In this lesson students will use the Penicillium chrysogenum fungus, which naturally produces the antibiotic penicillin, to illustrate the historical significance of naturally produced antibiotics. Students will co-culture P. chrysogenum and three species of bacteria to observe differences between penicillin-resistant and penicillin-sensitive bacteria. They will normalize both fungal and bacterial concentrations before co-cultivating, and quantify bacteria to determine antibiotic effect in liquid culture and on solid media. This will teach students about natural product antibiotics as well as experimental design and application.
This experiment allows students to practice several laboratory techniques, which are essential to careers in the biological sciences. They will cultivate and quantify microbiological organisms, practice accurate measurement techniques using several tools, conceptualize experimental design, and connect laboratory experiences to themselves and their understanding of medicine.
The activity requires one week (five days) of class time. Students should be organized into groups of two to four. Groups will collaborate to compile a complete data set for the experiments.
Figure 1. A) Penicillium lawn on PDA showing the ideal area of light green spores harvested and darkness of density of spores on swab. B) Spore suspension showing ideal density by color of LB/spores in 1.7ml micro centrifuge tube. C) Pure fungal cultures in 50ml LB showing ideal growth after four-day incubation. D) Bacterial culture tubes showing pipette tip from inoculation and ideal density after 24h incubation.
Figure 2. Vertical stripe co-cultivation showing placement of bacterial stripes perpendicular to fungal stripe on LB agar. The bacterial cultures show differential sensitivity to the penicillin diffused into the agar by the fungus.
Figure 3. Apparatus for filtering co-culture of bacteria and fungus consisting of coffee filters, conical tubes, a funnel and a rack capable of holding conical tubes upright. Bacteria will readily flow through the coffee filter and funnel into the conical tube. Fungus will remain trapped in the coffee filter.
Penicillium Fungus and Antibiotic Effect.
The discovery of penicillin revolutionized medicine to the degree that penicillin has been called the greatest contribution to medicine of the 20 th century. Before the discovery of penicillin and other antibiotics that followed, bacterial infections were common causes of death around the world. In the age before antibiotics, bacterial infections killed more people than heart disease and cancer combined. Today bacterial infections are considered trivial and are often treated with simple, inexpensive pills or ointments.
Penicillin was the first true antibiotic discovered in 1928 by Dr. Alexander Fleming. After returning from a vacation, Dr. Fleming noticed a greenish mold contaminating one of his bacteria plates. He observed that the bacteria would die within a zone around the mold leaving a clear ring. He isolated the mold in pure culture and determined that it was the common green bread mold, Penicillium. Many scientists would have dismissed this as a ruined experiment but Dr. Fleming was observant. His curiosity drove him to write a short scientific article on his observations.
Several years later, Fleming’s note caught the attention of Dr. Howard Florey, Dr. Norman Heatly, and Dr. Ernst Chain. These researchers conducted experiments to show that penicillin could be used to cure bacterial infections in a living organism (in vivo) and was suitable for use in mice and ultimately humans.
To understand what the early researchers might have witnessed, we can design an experiment to test the antibiotic effect of one species of Penicillium.
As a class, we will grow flasks of fungus in liquid cultures. We will test the fungus for penicillin antibiotic activity against three bacterial species with different levels of penicillin sensitivity: Staphylococcus epidermidis, Micrococcus luteus, and Enterobacter aerogenes. We will measure the concentration of bacteria in each flask after growing overnight with the fungus.
We can measure concentration of bacteria using a technique called optical density measurement (OD600). We will need to filter the fungus out of the culture, then we will compare the concentration of bacteria only cultures to each filtered co-culture.
- Practice accurate measurement techniques.
- Calculate dilutions.
- Observe bacterial sensitivity to penicillin.
- Contemplate designing experiments for accurate results.
Materials (recipes below)
Shared as a Class
- P. chrysogenum culture plate, grown 7 days on Potato Dextrose Agar (PDA)
- S. epidermidis LB agar plate, grown 48h at 30-37°C, can be stored at 4°C for £1 month
- M. luteus LB agar plate, grown 48h at 25-30°C, can be stored at 4°C for £1 month
- E. aerogenes LB agar plate, grown 24 h at 25-37°C, can be stored at 4°C for £1 month
- Orbital shaker
- 1x spectrophotometer (or colorimeter at 590-610nm)
- 1x sterile 2ml microcentrifuge tubes with 1.8ml LB (Miller)
- 3x sterile cotton tipped applicators
- 1x sterile box of p1000 tips
- 1x sterile box of p200 tips
- 6x cuvettes
- 6x sterile 125ml Erlenmeyer flasks with 25ml LB (Miller) each
- 1x LB agar plates
- 3x sterile 5ml culture tubes
- 3x sterile 50ml graduated cylinder or falcon tube
- 3x sterile 250ml Erlenmeyer flasks with 75ml 2xLB (Lennox) each
- 3x sterile 1ul inoculating loops or reusable metal loop
- 3x non-sterile 50ml Falcon collection tube
- 1x funnel (ensure spout fits in collection tube)
- 7x non-sterile coffee filters
(Working cultures need to be fresh)
- If starting from stock: Start fungal culture from stock onto potato dextrose agar (PDA) two weeks prior to activity. Incubate at 25°C for one week.
- Sub-culture fungus by spreading spores from primary (from stock) plates onto secondary PDA 1 week prior to activity and incubate at 25°C for one week (Figure 1a).
- Start bacterial cultures from glycerol stocks onto LB agar no more than two weeks and no less than two days before the co-cultivation experiment. Incubate M. luteus and S. epidermidis at 30°C for 48h and E. aerogenes at 30°C 24h. Bacterial culture plates may be stored at 4°C until activity.
- Serilize 25ml LB broth (Miller) in 125ml flasks (6 per group) and 75mL 2xLB broth (Lennox) in 250ml flasks (3 per group).
- Prepare 1 per group 2ml micro centrifuge tubes with 1.8ml LB (miller) broth
Penicillium (fungal) spore suspensions
(for Thermo Spectronic 200):
- Plug in and turn on. Wait until it shows “Remove cuvette and press ok…” Check that the spec is empty and press the ↵. Select OD600 from the menu options and press ↵ when the next screen appears
- Fill a cuvette with 900ml LB. Put it into the holder in the spec. Ensure that the clear windows of the cuvette are in line with the beam of light (facing left and right in a Spec 200). Close the lid and press the “0.00” button. Keep this cuvette for step 5. (Each cuvette has a slightly different effect on the measurement so make sure that you blank each cuvette in this way before use for the remainder of the experiments.)
- If using a WPA CO7500 colorimeter: Turn instrument on, place 900ul LB cuvette in the holder in the correct orientation (windows front to back) and press “Z” to blank.
- Label the 2ml centrifuge tubes containing 1.8ml LB broth with “SS” for spore suspension and your group number. (For example “SS1”) Dip a sterile cotton swab into the LB tube.
- Using sterile technique, collect spores from the P. chrysogenum plate by rubbing the LB broth soaked cotton swab on the plate. Collect an area roughly the size of your pinky finger. Press firmly while rubbing, but not so firmly to break the agar.
These plates can be shared among several groups. Select a fresh area of the culture for harvesting each time.
- Put the cotton swab with spores back in the SS tube and twirl it to mix in the spores. Continue about 10 seconds until the suspension looks cloudy, green, and homogenous.
- Make a 1/10 dilution in your cuvette from step 1:
- Pipette up and down to mix spore suspension.
- Pipette 100ml of your spore suspension into the same cuvette as in part 5a. Reseal and and set the SS tube aside.
- Pipette up and down to in the cuvette mix the dilution thoroughly.
- Make sure you leave all the suspension in the cuvette after mixing.
- Place the cuvette in the instrument and measure the absorbance.
- Make sure that the OD600 reading on the spec is above 1.0. If not, you need to add more spores from the plate to the 2mL tube
- Measure the spore concentration with the spec and record the result in Table 1 below (Absorbance values have no units like milliliters or grams)
- Remember the spec reading is 1/10 the Actual OD of the spores in your tube because you made a dilution. Multiply your Spec Readings by 10 to get the Actual OD.
Table 1: Spore suspension optical density data
P. chrysogenum sopre suspension
- We want to make sure we are working with the same starting concentrations for each of our experimental conditions. To do this we need to “normalize” the concentration of spores we add to each flask to the same amount. To normalize the volume of fungal spores you add to each culture, you will need to calculate the volume of SS we need to add to the culture flasks of LB broth for the ideal culture starting concentration.
a. For the dilution use the calculation equation C1 x V1=C2 x V2
(Actual OD in your 5ml tube) x (Volume of SS to add in ml) =
(0.05 ideal concentration in culture flask) x (25ml LB broth in culture flask)
c. (Volume of SS to add) = (0.05 x 25ml)/ (Actual OD in your 5mL tube)
d. Multiply answer by (1000ml /1ml) to convert to ml
V1 = (0.05 x 25ml)/13.6 = 0.092ml or 92μl
- Inoculate the liquid media flasks
- Mix the “SS” tube containing the spore suspension by inverting the tube 3 times.
- Loosen the foil on the flask before pipetting the volume from step d into three of the flasks containing 25ml LB.
- Use tape to label these flasks with the P. chrysogenum, your group number and initials, and the date.
(3 of your 6 flasks will be sterile LB that you will use it as a bacteria control in step 16.)
- Finally, we will use some of the remaining spores to make a vertical stripe on an LB agar plate.
- Label your LB agar plate “P. chrysogenum” with the date, and your initials
- Dip a new sterile cotton swab into the “SS” tube.
- Make one swipe across the center of the LB agar plate.
Culturing Bacteria (DAY 3)
- Inoculate 5ml LB broth (Miller) broth in a culture tube with each bacterial species.
- Label 3 culture tubes containing 5ml LB broth (Miller) each with your group number and the following:
- Using sterile technique, attach a sterile 20 – 200ul pipette tip to a p20 or p200 pipette.
- Incubate your tube with the rest of the class by shaking at 180 rpm overnight (until class the next day) at 30°C.
Co-cultivation (DAY 4)
On the 4 th day of the experiment, the cultures are ready to be co-cultivated. (bacteria added to fungus)
Potato dextrose broth medium NIKOORAEE
The Potato dextrose broth culture medium is used for the cultivation and counting of yeasts and molds, many microalgae and bacteria. Dextrose Agar potato is a product of Nikooraee company.
What is Brass Culture?
The potato dextrose agar medium is a general culture medium for yeast and mold. It can be supplemented with acid or antibiotics to prevent the growth of bacteria. The counting method is used when testing food, dairy and cosmetics. The USP cites potato dextrose agar as one of the recommended culture media for use in microbial counting experiments when testing unnecessary medicinal products. Dextrose potatoes are also used to stimulate spore formation (slide preparation), maintain cultures of specific dermatophytes, and differentiate atypical dermatophytes with pigment production. A general purpose watery medium for yeasts and molds that does not use agar, which is a solid.
Ingredients of PDB medium
Ingredients of Potato dextrose NIKOORAEE based on potato starch, potato extract and dextrose cause significant growth of fungi. Decreasing the pH of the medium to approximately 3.5 with sterile tartaric acid inhibits bacterial growth. However, it is important to avoid warming the medium after acidification as this will hydrolyze the agar and impair its ability to be strong. In general, to reduce material degradation, it is advisable to reduce the number of heat sterilization steps.
Instructions for preparation of potato dextrose broth medium
۱٫ Pour 24 grams of broth medium powder in 1 liter of distilled or purified water. If you want to use this medium by adding solid agar instead of 24 grams, you should use 39 grams per liter.
۲٫ While stirring repeatedly, heat it and simmer for 1 minute until the powder is completely dissolved. As a general guideline for the culture medium when your medium is completely dissolved when the solution boils.
۳٫ Autoclave at 121 ° C for 15 minutes.
۴- To change the reaction of potato dextrose agar medium to pH 3.5, cool the base to 45-50 ° C. Then add a decent amount of 10% sterile tartaric acid to the medium. Mix well. Do not reheat the agar-containing medium as the gelatinizing property of the agar may decrease or even disappear.
۵- Testing end-product samples for performance using stable and conventional control culture.
How to fill test tubes with potato dextrose broth
If you use test tubes or petri or any autoclavable container and similar tubing for cultivating your microorganism, it is best to add the potato dextrose into them before autoclaving, which will save a lot of time. Also, there is no possibility of contamination of the medium and container when transferring the autoclaved medium to the container.
۱- Wash and dry the test tubes first.
۲٫ Pour the warm potato dextrose NIKOORAEE medium (if it is broth , you can use it cool) to a third of the tube into the tube.
Close the pipe door using cotton or aluminum. If you are using aluminum, let the lid loose to allow heat and steam pressure into the tube when autoclaving.
۴- This stage is the autoclaving stage of the culture medium. The tubes should be positioned in an autoclave tank properly so that they do not overturn. You can put them into special pipes or even autoclavable containers like Besher the one with openings.
۵٫ After the autoclave is finished, place the tubes on a supine position, but beneath them, resting on a slope. However, this step is not required in the liquid-based Potato dextrose culture medium. But for solid media you should allow the medium to cool and solid.
For standard food, dairy and cosmetics, there are standard methods that can include disinfection before or without this stage. Note that too wet samples can increase bacterial growth.
Specimens can be placed on the potato dextrose culture medium in different ways. In the case of fragmented samples, you can place them with a forceps. For watery samples you can create small holes in the environment (by any means of sterile sharpening) and pour the liquid into these holes, but you can do this by circulating the liquid over the medium.
Place containers containing microorganisms on the potato dextrose broth medium at the desired temperature in light or dark conditions. If you are using petri, it is best to put them in a clean plastic and شnd fasten the door to close it to reduce environmental pollution.
Broth medium in tissue culture
The potato dextrose NIKOORAEE culture medium of Potito dextrose is based on a suitable medium for tissue culture of many plants. Many tissue culture and laboratory mass production companies are unaware of this environment and the possibility that it can be used as a public environment for many plants to grow. If you use Potito dextrose based media in tissue culture, you should adjust the pH of the plant before autoclaving.
Liquid media for mass production
The use of agar in the potato dextrose broth medium can pose many limitations, such as the transfer of material to seedlings and microorganisms in the liquid medium, but is restricted to the agar medium. It is also much easier to move microorganisms and seedlings into a new environment in a liquid environment and to add specific substances to them, but not in a solid environment.
Use of PDB medium for enzyme production and microorganism production
Many enzyme-producing plants and microbial products use potato dextrose-based media. The fluidity of this environment makes extraction of microbial products very easy and possible.
Can i grow bacteria on potato dextrose agar? - Biology
Produced by Jim Deacon
Institute of Cell and Molecular Biology, The University of Edinburgh
Temperature ranges of microorganisms
Microorganisms can be grouped into broad (but not very precise) categories, according to their temperature ranges for growth.
- Psychrophiles (cold-loving) can grow at 0 o C, and some even as low as -10 o C their upper limit is often about 25 o C.
- Mesophiles grow in the moderate temperature range, from about 20 o C (or lower) to 45 o C.
- Thermophiles are heat-loving, with an optimum growth temperature of 50 o or more, a maximum of up to 70 o C or more, and a minimum of about 20 o C.
- Hyperthermophiles have an optimum above 75 o C and thus can grow at the highest temperatures tolerated by any organism. An extreme example is the genus Pyrodictium, found on geothermally heated areas of the seabed. It has a temperature minimum of 82 o , optimum of 105 o and growth maximum of 110 o C.
It must be stressed that the temperature ranges for the groupings above are only approximate. For example, we would use different criteria to classify prokaryotes and eukaryotes. The upper temperature limit for growth of any thermophilic eukaryotic organism is about 62-65 o C. And the upper limit for any photosynthetic eukaryote is about 57 o - for the red alga Cyanidium caldarium, which grows around hot springs and has a temperature optimum of 45 o C. In contrast to this, some unicellular cyanobacteria can grow at up to 75 o C, and some non-photosynthetic prokaryotes can grow at 100 o C or more.
Below, we consider two major types of thermophile - the microbes that grow in geothermal sites, and those that grow in "self-heating" materials such as composts. However, some very recent reports suggest that these different types of environment can share some common organisms.
The study of extreme environments has considerable biotechnological potential. For example, the two thermophilic species Thermus aquaticus and Thermococcus litoralis are used as sources of the enzyme DNA polymerase, for the polymerase chain reaction (PCR) in DNA fingerprinting, etc. The enzymes from these organisms are stable at relatively high temperatures, which is necessary for the PCR process which involves cycles of heating to break the hydrogen bonds in DNA and leave single strands that can be copied repeatedly. Another thermophile, Bacillus stearothermophilus (temperature maximum 75 o C) has been grown commercially to obtain the enzymes used in 'biological' washing powders.
Hot springs and geothermal vents are found in several parts of the world, but the largest single concentration is in Yellowstone National Park, USA. The images below show how some thermophilic prokaryotes (bacteria and archaea) are specially adapted to grow in these environments. In each case we find a zonation of microorganisms according to their temperature optima. Often these organisms are coloured, due to the presence of photosynthetic pigments (blue-green of cyanobacteria, red of red algae or purple bacteria) or carotenoid pigments (yellows and browns of some archaea).
MT Madigan, JM Martinko & J Parker (1997) Brock Biology of Microorganisms. Eighth edition.. Prentice Hall. (see http://www.prenhall.com/brock/ )
MT Madigan & BL Marrs (1997) Punishing Environments: Extremophiles. Scientific American issue 4 ( http://www.sciam.com/0497issue/0497marrs.html )
M.W.W. Adams and R.M. Kelly. (1995) Enzymes Isolated from Microorganisms That Grow in Extreme Environments. Chemical and Engineering News 73, 32-42.
Extremophiles. Special issue of Federation of European Microbiological Societies (FEMS) Microbiology Reviews 18, Nos. 2-3 May 1996.
K. O.Stetter (1996) Hyperthermophiles in the History of Life. in Evolution of Hydrothermal Ecosystems on Earth (and Mars?). Edited by GR Bock & JA Goode. John Wiley & Sons.
A compost consists of any readily degradable organic matter that is kept in a heap with sufficient mineral nutrients (e.g. nitrogen) and sufficient aeration to enable rapid microbial growth. The most familiar example is the garden compost heap, but more important examples are the composting plants used to process municipal wastes, and the composts used for commercial mushroom production.
The typical composting process is illustrated in Figure E below. There is an initial phase of rapid microbial growth (a) on the most readily available sugars and amino acids. This phase is initiated by mesophilic organisms, which generate heat by their metabolism and raise the temperature to a point where their own activities are suppressed. Then a few thermophilic fungi (e.g. Rhizomucor pusillus, Figure F) and several thermophilic bacteria (e.g. Bacillus stearothermophilus) continue the process, raising the temperature of the material to 70-80 o C within a few days. This peak heating phase (b) has a profound effect on the microbial population, because it destroys or inactivates all the mesophilic organisms (and the initial thermophilic fungi such as Rhizomucor pusillus) and leads to a prolonged high-temperature phase that favours other thermophilic species.
Figure E. Changes in temperature (solid line) and populations of mesophilic fungi (broken line) and thermophilic fungi (dotted line) in a wheat straw compost. Based on data in Chang & Hudson, 1967. The left axis shows fungal populations (logarithm of colony forming units per gram of compost plated onto agar) the right axis shows temperature in the centre of the compost. Stages a-d are referred to in the text.
Eventually the temperature declines and mesophilic organisms then recolonise the compost and displace the thermophiles (d in Fig. E). However, some heat-tolerant species such as Aspergillus fumigatus can continue to grow. This fungus can grow at temperatures ranging from 12 o to about 52-55 o . Strictly speaking, it is not a thermophile because its temperature optimum is below 50 o , but it is a very common and important member of the high-temperature compost community.
All the thermophilic fungi shown below were obtained by plating small particles of garden compost on potato-dextose agar containing antibacterial agents (streptomycin plus chlortetracycline) at 45 o C.
Figure F. Rhizomucor pusillus. Typical grey coloured colony on a plate of potato-dextrose agar at 45 o C (left-hand image). This fungus produces abundant "fluffy" aerial hyphae and spore-bearing stalks (sporangiophores) which are branched (centre and right images) and have sporangia at the tips of the branches. The delicate sporangial walls break to release numerous spores, leaving only a central bulbous region (the columella, c) and remains of the sporangial wall (arrowhead in right-hand image).
With a temperature range of 20-55 o C, this fungus is a typical early coloniser of composts, exploiting simple sugars, amino acids etc. that are present initially in the plant material. It is inactivated during peak-heating, and it does not recolonise afterwards.
Figure G. Humicola (or Thermomyces) lanuginosus. Colonies growing on potato-dextrose agar (top left) and malt extract agar (top right) at 45 o C. This fungus produces single spores by a balloon-like swelling process at the tips of short hyphal branches (bottom, left). At maturity (bottom right) the spores have brown, ornamented walls.
H. lanuginosus grows from 30 to 52-55 o C. It is extremely common in all types of self-heating material and also in birds' nests and sun-heated soils. It colonises composts after peak-heating and persists throughout the high-temperature phase. However, it cannot degrade cellulose and it seems to live as a commensal with cellulose-decomposing species, sharing some of the sugars released from the plant cell walls by their cellulolytic activities.
Figure H. Thermoascus aurantiacus colony (left) growing on malt-extract agar at 45 o C. The orange-brown colour is caused by the presence of many small (about 1 millimetre) fruiting bodies (ascocarps), which are seen at higher magnification (top right). These ascocarps are closed bodies, termed cleistothecia, containing many asci, each with 8 ascospores. The cleistothecia and the ascus walls break down at maturity to release the ascospores. Four asci containing ascospores are shown in the composite image at bottom right. They were released when a cleistothecium was crushed on a slide, and they show ascospores in various stages of maturity - the brown spores are nearly mature.
This fungus grows from about 25 to 55 o C and is a vigorous cellulose degrader.
Figure I. Paecilomyces species growing on malt extract agar at 45 o C (left image). The yellow-buff colour of the colony is caused by the presence of asexual sporing structures on the aerial hyphae. The sporing stages of the genus Paecilomyces (right-hand image) superficially resemble those of Penicillium, because the spores (conidia, c) are formed from flask-shaped cells (phialides, p) borne at the tips of short, brush-like branching structures. But the branching pattern of these "brushes" is less regular than in Penicillium .
Figure J. Aspergillus fumigatus. This common fungus of composts and mouldy grain has a grey-green colour on agar plates (left image), in contrast to the brighter green colour of several other Aspergillus species. The typical asexual sporing stage of Aspergillus consists of a spore-bearing hypha (conidiophore, centre image) which swells into a vesicle at the tip, and the vesicle bears flask-shaped cells (phialides) that produce the spores (conidia). In A. fumigatus the vesicle typically is club-shaped, the phialides arise only from the upper part of the vesicle, and the phialides all point upwards. Together with the grey-green colour and temperature range of about 12-52 o C, these features distinguish A. fumigatus from all other Aspergillus species.
A. fumigatus is an extremely common, interesting and dangerous fungus because of its nutritional opportunism. It is strongly cellulolytic, but it also can grow on hydrocarbons in aviation kerosene, and it can enter the lungs as inhaled spores, causing allergies or growing in the lung cavities, causing aspergillomas (see Airborne Microorganisms ). Its ability to grow readily at 37 o C has made this fungus a significant problem in operating theatres, where it can establish infections of the internal organs via surgical wounds, especially during transplant surgery when the patient's immune system is suppressed.
Although the thermophilic fungi play a major role in degrading cellulose and other major polymers in composts, the activities of bacteria also are important. Two recent discoveries highlight this point.
- In 1996 it was reported that composts of many different types (garden and kitchen wastes, sewage sludge, industrial composting systems) contain high numbers of bacteria of the genus Thermus which grow on organic substrates at temperatures from 40-80 o C, with optimum growth between 65 and 75 o C. The numbers were as high as 10 7 to 10 10 per gram dry weight of compost. Spore-forming Bacillus species were also found, but they were unable to grow above 70 o C. Thus, it seems that Thermus species, previously known only from geothermal sites, have probably adapted to the hot-compost system and play a major role in the peak-heating phase. [T. Beffa et al., 1996. Applied & Environmental Microbiology62, 1723-1727].
- Also in 1996, a number of autotrophic (self-feeding) bacteria were isolated from composts. These non-sporing bacteria grew at 60-80 o C, with optima of 70-75 o C, and closely resembled Hydrogenobacter strains that previously were know only from geothermal sites. They obtain their energy by oxidising sulphur or hydrogen, and synthesise their organic matter from CO2. [T. Beffa et al., 1996. Archives of Microbiology165, 34-40]
There is much to be learned about the interactions of microorganisms in composting systems. Work in this field is driven largely by commercial needs to produce composts for high mushroom yields (Agaricus species) and for rapid, efficient processing of municipal (domestic) and industrial wastes.
In contrast to the typical "natural" composting sequence (Figure E), commercial mushroom composts are produced by a truncated, two-phase process, designed to minimise the loss of cellulosic materials that Agaricus can use for growth. Phase I involves peak-heating of straw compost to 70-80 o C for several days. Then the compost is pasteurised at 70 o C and held at about 45 o C for a further few days (Phase II). Finally, the temperature is lowered and the compost is inoculated with Agaricus. Both of the preliminary phases are essential for high mushroom yields. Recent work suggests that the thermophilic fungus Scytalidium thermophilum becomes dominant in phase II and its presence can almost double the mushroom yield [G. Straatsma et al., 1995. Canadian Journal of Botany 73, S1019-1024]. The reason for this is still unknown.
Other work has shown that Agaricus bisporus (the commercial mushroom) can use either living or heat-killed bacteria as its sole source of nitrogen for growth. Like many other members of the fungal group basidiomycota, this fungus does not thrive on inorganic nitrogen sources such as ammonium or nitrate, but readily utilises organic nitrogen, which it can degrade by releasing protease enzymes. Thus, an initial high bacterial activity in the compost might provide the fungus with its favoured nitrogen source.
Can i grow bacteria on potato dextrose agar? - Biology
Antibiotics are the organic secretion produced by micro &ndash organisms, which in low concentrations are antagonistic to the growth of other micro &ndash organisms (mostly pathogens). Antibiotics have proved very useful in combating several bacterial diseases in man an animal. Antibiotics are commonly obtained from actinomycetes and some eubacteria. Some of the important antibiotics are streptomycin, Aureomycin, teramycin, Chloromycetin, erythromycin, neomycin etc.
Soil is a natural medium that harbors several types of micro &ndash organism .These micro- organisms can be frown on culture media. The effect of different types of antibiotics can be studied on the growth of micro - organism growing in culture medium. This is an important subject, therefore the study of effect of antibiotics on micro - organism has been taken for the present project.
Objective of Project
The aim of this project is to study the effect of antibiotics on micro &ndash organisms.
To study the effect of antibiotics on micro-organisms
Potato ,agar, dextrose , distilled water , four different types of antibiotics (such as penicillin , streptomycin , aureomycin , teramycin ) , syringe , oven sterilized petridish , flasks , beakers , pipettes , garden soil , glass marker pen , etc
A. Preparation of culture Medium
1. Potato Dextrose Agar (PDA) Medium.
Take 200 g of peeled potato chips. Boil them with 500 ml of water in a beaker for 15 minutes.
Squeeze the potato pulp thus obtained through a muslin cloth and keep it in a flask.
Take 20 g of agar in a beaker and warm it with 500 ml of water .
Mix both the solution of potato and agar and add 20 g dextrose to it.
Thus one litre of PDA medium is prepared.
Autoclave the medium at 15 pounds pressure for 15 minutes.
B. Effects of antibiotics on soil micro0 organisms
i) Take 2 g of soil and dissolve it in 10 ml of water in a beaker. Let the soil particle settle down.
ii) Take 5 oven sterilized petridishes and pour 1 ml of soil suspension in each of the plates. Now pour 1ml of the four antibiotics separately in four petridishes with the help of syringe, and mark them with marker pen. Leave the fifth petridish without antibiotic to serve as control.
iii) Pour PDA in each of the petridishes and mix the suspension by rotating the petridishes. Leave the petridishes undisturbed at a warm place
The effect of different antibiotics on the micro &ndash organisms can be assessed by counting the number and size of the colonies growing in the petridishes.
Pencillin and Terramycin was most effective antibiotic against microorganism in soil.
Sabouraud agar or Sabouraud dextrose agar (SDA) is a type of agar growth medium containing peptones.  It is used to cultivate dermatophytes and other types of fungi, and can also grow filamentous bacteria such as Nocardia.    It has utility for research and clinical care.
It was created by, and is named after, Raymond Sabouraud in 1892. In 1977 the formulation was adjusted by Chester W. Emmons when the pH level was brought closer to the neutral range and the dextrose concentration lowered to support the growth of other microorganisms. The acidic pH (5.6) of traditional Sabouraud agar inhibits bacterial growth. 
Sabouraud agar is commercially available and typically contains: 
Clinical laboratories can use this growth medium to diagnose and further speciate fungal infections, allowing medical professionals to provide appropriate treatment with antifungal medications. Histoplasma and other fungal causes of atypical pneumonia can be grown on this medium.
- ^"Omnipresence of Microorganisms in the Environment". Archived from the original on 2008-10-06 . Retrieved 2008-10-24 .
- Sandven P Lassen J (November 1999). "Importance of selective media for recovery of yeasts from clinical specimens". Journal of Clinical Microbiology. 37 (11): 3731–2. PMC85742 . PMID10523586.
- Guinea J Peláez T Alcalá L Bouza E (December 2005). "Evaluation of Czapeck agar and Sabouraud dextrose agar for the culture of airborne Aspergillus conidia". Diagnostic Microbiology and Infectious Disease. 53 (4): 333–4. doi:10.1016/j.diagmicrobio.2005.07.002. PMID16263232.
- ^About Modified Sabouraud Agar
- Hare, Janelle M. (9 December 2012). "15. Sabouraud agar for fungal growth". In Gupta, Vijai Kumar Tuohy, Maria G. Ayyachamy, Manimaran Turner, Kevin M. O’Donovan, Anthonia (eds.). Laboratory Protocols in Fungal Biology: Current Methods in Fungal Biology. Springer. p. 212. ISBN978-1-4614-2355-3 .
- ^University of Sydney, Recipes.
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