PSEUDOMONAS AGAR BASE

Code: CM0559

For the selective isolation of Pseudomonas species when supplemented with SR0102 or SR0103.

* Adjusted as required to meet performance standards

PSEUDOMONAS CN SELECTIVE SUPPLEMENT

Code: SR0102

PSEUDOMONAS CFC SELECTIVE AGAR SUPPLEMENT

Code: SR0103

Directions
To Prepare the Agar Base
Suspend 24.2g of the agar base, in 500ml of distilled water. Add 5ml of glycerol. Bring to the boil to dissolve completely, sterilise by autoclaving at 121°C for 15 minutes. Allow the medium to cool to 50°C.
To Prepare Pseudomonas CN Agar
To 500ml of agar base cooled to 50°C add the contents of 1 vial of Pseudomonas CN Supplement (SR0102) rehydrated as directed. Mix well and pour into sterile Petri dishes.
To Prepare Pseudomonas CFC Agar
To 500ml of agar base cooled to 50°C add the contents of 1 vial of Pseudomonas CFC Supplement (SR0103) rehydrated as directed. Mix well and pour into sterile Petri dishes.

Description
Pseudomonas Agar Base is designed so that by the addition of the appropriate supplement (SR0102 or SR0103) the medium becomes selective for Pseudomonas aeruginosa or Pseudomonas spp. generally. The base medium is a modification of King’s A Medium¹ in which magnesium chloride and potassium sulphate are present to enhance pigment production.

Pseudomonas CN Supplement (SR0102) is recommended for the selective isolation of Pseudomonas aeruginosa. The formula of the supplement was described by Goto and Enomoto² following the demonstration of cetrimide as a selective agent by Lowbury and Collins³. Goto and Enomoto showed that addition of nalidixic acid at 15µg/ml, while at the same time reducing the cetrimide content to 200mg, improved performance. The medium gave better recovery of Pseudomonas aeruginosa with enhanced pigment formation whilst strongly suppressing Klebsiella, Proteus and Providencia spp., the latter organisms being the troublesome contaminants of conventional cetrimide media.

Pseudomonas CFC Supplement (SR0103) is recommended for the selective isolation of Pseudomonas spp. generally. Mead and Adams⁴ showed that reducing the cetrimide content to 10mg/ml allowed the growth of all pigmented and non- pigmented psychrophilic pseudomonads. To improve its selective action they added cephaloridine 50µg/ml) and fucidin (10µg/ml). This combination of agents gave a new and more specific medium for isolating pseudomonads from chilled foods and processing plants. Incubation should be carried out at 25-30°C for 48 hours.

Considerable importance is given to detection of Burkholderia cepacia (formerly Pseudomonas cepacia) in water systems, particularly where the water is to be used for the preparation of pharmaceuticals and cosmetics⁵. The organism is resistant to many commonly-used disinfectants. Burkholderia cepacia has emerged as an important opportunistic pathogen in urinary, abdominal, respiratory and other infections.

Growth characteristics of Pseudomonas species on Oxoid Pseudomonas Agar Base with Supplements.

Incubation carried out at 30°C and plates read after 18 hours incubation.

Technique
Clinical Specimens for Pseudomonas aeruginosa Investigations
1. Prepare Pseudomonas CN Medium as directed.
2. Pour plates and dry the surface.
3. Swab a large inoculum over half the area of the plate.
4. Using a sterile loop, streak out the inoculum over the remainder of the plate to obtain isolated colonies.
5. Incubate at 35°C and examine after 24 and 48 hours, using both white and ultraviolet light.

Food, Water and Environmental Samples for Pseudomonads
1. Prepare Pseudomonas CFC Medium as directed.
2. Pour plates and dry the surface.
3. Prepare food samples by diluting 1 in 5 or 1 in 10 with 1% (w/v) sterile Peptone Water (CM0009) and homogenise in a `Stomacher’ or a laboratory blender.
4. Pipette 0.5 or 1ml of the homogenate on to the plate and spread over the surface with a sterile glass spreader. Inoculate water and swab samples directly on the surface of the medium.
5. Incubate at 25°C and examine after 24 and 48 hours, using both white and ultraviolet light.

技术
铜绿假单胞菌临床标本研究
1. 按照指示制备假单胞菌 CN 培养基。
2. 倒入盘子并擦干表面。
3. 将一个大的接种物擦拭到平板的一半区域。
4. 使用无菌环，将接种物划线覆盖板的其余部分，以获得分离的菌落。
5. 在 35°C 下孵育，并在 24 和 48 小时后使用白光和紫外线进行检查。

假单胞菌的食物、水和环境样品
1. 按照指示制备假单胞菌 CFC 培养基。
2. 倒入盘子并擦干表面。
3. 用 1% (w/v) 无菌蛋白胨水 (CM0009) 稀释 1 比 5 或 1 比 10 来制备食品样品，并在“Stomacher”或实验室搅拌器中均质化。
4. 将 0.5 或 1ml 匀浆移液到板上，并用无菌玻璃散布器涂抹在表面上。直接在培养基表面接种水和拭子样本。
5. 在 25°C 下孵育，并在 24 和 48 小时后使用白光和紫外线进行检查。

Colonial Appearance
Growth on CN or CFC Medium is usually limited to Pseudomonas spp. but some members of the family Enterobacteriaceae may also be present. The presence of blue-green or brown pigmentation, or fluorescence may be taken as presumptive evidence of Pseudomonas spp. but further tests must be carried out to confirm the identity of the organism.
Stanbridge and Board⁶ modified CFC Medium to differentiate pseudomonads from Enterobacteriaceae developing on beef steaks packaged in modified atmospheres. Arginine 1% w/v and phenol red 0.002% w/v were added to the medium.
Pseudomonads produce ammonia from the arginine and colonies may be distinguished by a pink coloration.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared medium at 2-8°C.

Appearance
Dehydrated medium: Straw coloured, free-flowing powder
Prepared medium: Straw coloured gel

殖民地外观
CN 或 CFC 培养基上的生长通常仅限于假单胞菌属。 但也可能存在肠杆菌科的一些成员。 蓝绿色或棕色色素沉着或荧光的存在可作为假单胞菌属的推定证据。 但必须进行进一步的测试以确认生物体的身份。
Stanbridge 和 Board6 改良了 CFC 培养基，以区分在气调包装的牛排上生长的假单胞菌和肠杆菌科细菌。 向培养基中加入 1% w/v 精氨酸和 0.002% w/v 酚红。
假单胞菌从精氨酸中产生氨，菌落可以通过粉红色来区分。

储存条件和保质期
将脱水培养基储存在 10-30°C 并在标签上的有效期之前使用。
将准备好的培养基储存在 2-8°C。

外貌
脱水介质：稻草色、自由流动的粉末
制备培养基：稻草色凝胶

Quality control

* This organism is available as a Culti-Loop®

Precautions
Fresh media should be prepared as required. Molten agar should not be kept longer than 4 hours. Medium should not be stored and remelted. If swarming colonies of Proteus species are a problem in food samples then the incubation temperature can be lowered to 20°C for a period of 3-5 days. Chilled foods may carry a wide range of pseudomonads and the colonies on CFC Medium, incubated at lower temperatures, may be Pseudomonas fluorescens or Pseudomonas putida as well as Pseudomonas aeruginosa. Aeromonas species will also appear as pink/brown colonies, particularly from fish products.

防范措施
应根据需要准备新鲜培养基。 熔化的琼脂不应保存超过 4 小时。 培养基不应储存和重熔。 如果变形杆菌属的菌落在食品样品中出现问题，则可以将孵育温度降低至 20°C，持续 3-5 天。 冷藏食品可能携带多种假单胞菌，CFC 培养基上的菌落在较低温度下培养，可能是荧光假单胞菌或恶臭假单胞菌以及铜绿假单胞菌。 气单胞菌也将出现粉红色/棕色菌落，尤其是来自鱼产品的菌落。

References
1. King E.O., Ward M.K. and Raney D.E. (1954) J. Lab. & Clin. Med. 44. 301-307.
2. Goto S. and Enomoto S. (1970) Jap. J. Microbiol. 14. 65-72.
3. Lowbury E.J. and Collins A.G. (1955) J. Clin. Path. 8. 47-48.
4. Mead G.C. and Adams B.W. (1977) Br. Poult. Sci. 18. 661-667.
5. Geftic S.G., Heymann H. and Adair F.W. (1970) App. & Environmental Microbiol. 37. 505-510.
6. Stanbridge L.H. and Board R.G. (1994) Lett. Appl. Microbiol. 18. 327-328.