microorganisms present while they are being transported to the laboratory for cultures. because it is unknown whether the pathogens are anaerobic (living without oxygen) or aerobic (living with oxygen), blood is collected to test for both. the aerobic bottle is filled first, and then the anaerobic bottle is filled. however, if the collection is performed using a syringe, the anaerobic bottle is filled first. if a butterfly collection kit is used, the aerobic bottle is filled first, so that any air in the tubing is released into the oxygen-containing bottle. specially designed blood culture collection bottles eliminate the need for either the syringe or butterfly collection method. these specially designed bottles have long necks that fit into the evacuated tubes holders that are use for regular venipuncture collection. these bottles also allow for collection of other blood specimens via evacuated tubes, to be collected without additional venipuncture. the amount of blood that is collected is critical for the optimal recovery of microorganisms. up to 10ml of blood is typical, but can vary according to the recommends of the manufacturer of the collection bottle. collection from infants and children are 1 to 5 ml. if too little blood is collected, the ratio of blood-to-nutrient broth will inhibit the growth of microorganisms. if too much blood is collected, there is the risk of a hospital-induced anemia and the ratio of blood-to-nutrient broth will tilt in the opposite direction, which also is not conductive to optimal growth. the bottles are then incubated in specialized units for 24 hours before a lab technician studies and/or tests it. this step allows the very small numbers of bacteria (potentially 1 or 2 organisms) to multiply to a level which is sufficient for identification +/-antibiotic resistance testing. modern blood culture bottles have an indicator in the base which changes color in the presence of bacterial growth and can be read automatically by machine. (for this reason the barcoded stickers found on these bottles should not be removed as they are used by the laboratory's automated systems.) taking blood samples from animals blood samples from living laboratory animals may be collected using following methods: blood collection not requiring anesthesia: saphenous vein (rat, mice, guinea pig) dorsal pedal vein (rat, mice) blood collection requiring anesthesia (local/general anesthesia): tail vein (rat, mice) tail snip (mice) orbital sinus (rat, mice) jugular vein (rat, mice) temporary cannula (rat, mice) blood vessel cannulation (guinea pig, ferret) tarsal vein (guinea pig) marginal ear vein or artery (rabbit) terminal procedure: cardiac puncture (rat, mice, guinea pig, rabbit, ferret) orbital sinus (rat, mice) posterior vena cava (rat, mice) the volume of the blood sample collection is very important in experimental animals. all nonterminal blood collection without replacement of fluids is limited up to 10% of total circulating blood volume in healthy, normal, adult animals on a single occasion and collection may be repeated after three to four weeks. in case repeated blood samples are required at short intervals, a maximum of 0.6 ml/kg/day or 1.0% of an animal's total blood volume can be removed every 24 hours. the estimated blood volume in adult animals is 55 to 70 ml/kg body weight. care should be taken for older and obese animals. if blood collection volume exceeds more than 10% of total blood volume, fluid replacement may be required. lactated ringer's solution (lrs) is recommended as the best fluid replacement by national institutes of health (nih). if the volume of blood collection exceeds more than 30% of the total circulatory blood volume, adequate care should be taken so that the animal does not develop hypovolemia. blood alcohol tests it is generally not advisable to use isopropyl alcohol to cleanse the venipuncture site when obtaining a specimen for a blood alcohol test. this has been related largely to the potential legal implications associated with the use of alcohol-based cleaners that could theoretically impact analysis. numerous police alcohol collection kits have been marketed that incorporate a sodium fluoride/potassium oxalate preservative and non-alcohol-based cleansing agents to ensure proper collection. using soap and hot water or a povidone-iodine swab are advisable alternatives to isopropyl alcohol in this case.